extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Null mutation of chloride channel 7 (Clcn7) impairs dental root formation but does not affect enamel mineralization

ClC-7, located in late endosomes and lysosomes, is critical for the function of osteoclasts. Secretion of Cl− by the ruffled border of osteoclasts enables H+ secretion by v-H+-ATPases to dissolve bone mineral. Mice lacking ClC-7 show altered lysosomal function that leads to severe lysosomal storage. Maturation ameloblasts are epithelial cells with a ruffled border that secrete Cl− as well as endocytose and digest large quantities of enamel matrix proteins during formation of dental enamel. We tested the hypothesis that ClC-7 in maturation ameloblasts is required for intracellular digestion of matrix fragments to complete enamel mineralization. Craniofacial bones and developing teeth in Clcn7-/- mice were examined by micro-CT, immunohistochemistry, quantified histomorphometry and electron microscopy. Osteoclasts and ameloblasts in wild-type mice stained intensely with anti-ClC-7 antibody but not in Clcn7-/- mice. Craniofacial bones in Clcn7-/- mice were severely osteopetrotic and contained 1.4- to 1.6-fold more bone volume, which was less mineralized than the wild-type littermates. In Clcn7-/- mice maturation ameloblasts and osteoclasts highly expressed Ae2 as in wild-type mice. However, teeth failed to erupt, incisors were much shorter and roots were disfigured. Molars formed a normal dental crown. In compacted teeth, dentin was slightly less mineralized, enamel did not retain a matrix and mineralized fairly normal. We concluded that ClC-7 is essential for osteoclasts to resorb craniofacial bones to enable tooth eruption and root development. Disruption of Clcn7 reduces bone and dentin mineral density but does not affect enamel mineralization.

Introduction
Maturation ameloblasts, epithelial cells that deposit and mineralize dental enamel, have some structural and functional similarities with bone-resorbing osteoclasts. Osteoclasts and ameloblasts both have a ruffled border facing a calcified extracellular matrix and both secrete enzymes to degrade and remove this matrix (Josephsen and Fejerskov 1977;Salama et al. 1989Salama et al. , 1990Zhao and Patrick Ross 2007). Both also have a similar pH-regulatory machinery including carbonic anhydrase-2, anion exchanger-2 (Ae2), v-H + -ATPase (Josephsen et al. 2010;Lin et al. 1994) and sodium bicarbonate cotransporter-1(Nbce1) (Jalali et al. 2014;Josephsen et al. 2010). Osteoclasts use this pH regulatory machinery to produce and secrete protons at their ruffled border with a central role for v-H + -ATPase. Maturation ameloblasts that mineralize dental enamel also express v-H + -ATPase and it has been proposed that these cells secrete protons to prevent precocious mineralization at the enamel surface that would otherwise inhibit deeper layers of enamel to mineralize (Josephsen et al. 2010). However, mineralization of enamel in mice with null mutation of Tcirg1 (the osteoclast-specific subunit of the v-H + -ATPase proton pump in the ruffled border), was not different from wild-type enamel, suggesting that the v-H + -ATPase detected in maturation ameloblasts was another type of v-H + -ATPase involved in acidification of intracellular vesicles and trafficking rather than a plasma membraneassociated proton pump to secrete protons as in osteoclasts (Bronckers et al. 2012).
ClC-7 belongs to the CLC family of chloride channels and transporters, which consists in nine mammalian members with diverse physiological roles (Stauber et al. 2012). The CLC family comprises both plasma membrane-localized chloride channels and chloride-proton exchangers that reside predominantly in membranes of compartments of the endocytic pathway (Jentsch 2008;Stauber et al. 2012). ClC-7, a Cl − / H + antiporter (Leisle et al. 2011) and its β-subunit Ostm1 localize to lysosomes of all cells and additionally reside at the ruffled border membrane of osteoclasts (Kornak et al. 2001;Lange et al. 2006). ClC-7 in parallel to the v-type H + -ATPase is important for the acidification of the resorption lacuna (Kornak et al. 2001). In contrast to its effect on the pH in the resorption lacuna, the lysosomal pH is not changed in cells lacking ClC-7 (Kasper et al. 2005;Steinberg et al. 2010); lysosomal acidification is perhaps enabled by cation efflux (Steinberg et al. 2010). ClC-7 seems rather involved in Cl − accumulation in lysosomal vesicles (Weinert et al. 2010).
Disruption of the Clcn7 gene in mice results in severe osteopetrosis in the long bones of the extremities, shorter stature and splenomegaly (Kornak et al. 2001). Failure of teeth to erupt has also been reported for Clcn7 -/mice but the description was not very detailed (Kasper et al. 2005;Kornak et al. 2001;Wen et al. 2015).
Ameloblasts secrete proteases as MMP20 and KLK4 to degrade most of the enamel matrix followed by endocytosis and digestion of at least a portion of the peptide fragments, particularly during maturation stage. Retention of the enamel matrix and incomplete enamel mineralization happen in mice with null mutation of Mmp20, Klk4 or Sppl2a (an intramembranous protease located in lysosomes) (Bartlett et al. 2004;Bronckers et al. 2013;Simmer et al. 2009). Transcripts for nine CLC members have been identified in mouse ameloblasts, in the maturation stage most abundantly Clcn7 and ClC-7 protein was immunolocated in ameloblast vesicles (Lacruz et al. 2013). It was unknown whether disruption of Clcn7 affects formation of enamel and dentin or changes the expression of other important chloride exchangers.
In view of the heterogeneity of osteoclasts, it was also unknown whether in Clcn7 -/mice all bony structures are affected. Osteoclasts in the craniofacial bones differ in some aspects from those in long bones ). In pycnodysostosis, the Ctsk mutation leads to sclerosis of the long bones but not in craniofacial bones (Gowen et al. 1999;Saftig et al. 2000). Null mutation of Ae2a,b causes osteopetrosis in long bones, not in craniofacial bones and disrupts formation of dental enamel ).
In this study, we address two questions: (1) Is a functional ClC-7 required for normal formation of teeth, in particular for different stages of amelogenesis and root formation? (2) Does disruption of Clcn7 also affect the function of osteoclasts in craniofacial bones resulting in osteopetrosis? We examine the functional relevance of ClC-7 for the development of teeth and craniofacial bones by studying changes in the structure of dental and bone tissues in Clcn7 -/mice (Kornak et al. 2001;Neutzsky-Wulff et al. 2008).

Mice
Heads and bones were obtained from six mice of the genetically modified Clcn7 mouse strain and six of their wild-type littermates (courtesy Dr. T.J.Jentsch, Berlin, Germany). The Clcn7 null mouse strain was generated by deletion of exons 3-7 in the Clcn7 gene, which completely abolished expression of ClC-7 (construct C7A) as described by Kornak et al. (2001). Pups were fed with a soft (gel) diet each day after weaning until sacrificed at ages post-natal days 22 or 23. Three pairs of the mice were used for ultrastructural studies and another three pairs were used for micro-CT analysis, immunolocalization and histomorphometric analysis. All procedures were approved by the Committee for Animal Health and Animal Care of the VU-University and FMP/MDC in Berlin, Germany, according to national standards.

Tissue fixation, embedding and sectioning
Mice were perfused first with phosphate-buffered saline (PBS) and then with 4 % formaldehyde in PBS. Heads and bones were collected and postfixed in 4 % formaldehyde in PBS overnight at 4°C. From each group of 3 mice, mineralized tissues were scanned for micro-CT analysis. The samples were then decalcified in 4.18 % EDTA+0.8 % formalin at pH 7.2 for 4 weeks at 4°C, rinsed with phosphate buffer, embedded in paraffin and serially sectioned into 6-μm-thick sections for immunostaining and staining with hematoxylineosin (HE). For ultrastructural studies, mouse heads of Clcn7 -/ mice and wild-type controls were fixed in 1 % glutaraldehyde and 4 % formaldehyde in 0.1 M sodium cacodylate buffer pH 7.3 for 1-2 weeks. Then, the tissues were demineralized in 4.2 % EDTA+0.8 % formaline for 2 weeks. After postfixation in 1 % OsO 4 for 1 h, the tissues were dehydrated in ethanol and embedded in epoxy resin (LX112).

Immunohistochemistry
EDTA-decalcified, paraffin-embedded tissue sections were immunostained with affinity purified rabbit anti-human ClC-7 (Abcam, catalogue ab31264) and rabbit anti-Ae2 (courtesy Dr. S. Kellokumpu, University of Oulu, Finland). According to the manufacturer, anti-ClC-7 antibody was raised against a synthetic peptide-conjugate to KLH from within amino acid residues 750 to C-terminus of the human ClC-7, with a predicted 94 % identity with mouse, rat and rabbit. Sections were deparaffinized in xylene, rehydrated in ethanol and washed in Tris-buffered saline (TBS). Antigen retrieval was carried out for both antibodies by incubation in 10 mM citrate buffer (pH 6.0) overnight at 60°C. Nonspecific staining of the tissues was blocked by 30 min incubation with normal goat sera. Sections were then incubated with primary antibodies (1:200 dilution for ClC-7 antibody, 1:100 dilution for the anti-Ae2 antibody) or non-immune rabbit IgG (negative controls) overnight at 4°C. After washing in TBS, sections were incubated with goat anti-rabbit-IgG-polymer conjugated with peroxidase (EnVision kit; Dako Cytomation, Glostrup, Denmark) for 1 h at ambient temperature Schaller et al. 2004), washed and the peroxidase visualized using DAB (EnVision kit), counterstained with hematoxylin, dehydrated and mounted in Depex.

Microcomputer tomography (micro-CT)
The heads and tibia from three Clcn7 -/mice and three wildtype littermates were scanned under the same conditions at a resolution of 6 μm voxels using a μCT-40 high-resolution scanner (Scanco Medical, Bassersdorf, Switzerland). After scanning, 3-D computer reconstructions were made of the incisors, first molars, calvarial and jaw bones to detect structural changes in mineralized tissues in Clcn7 -/mice. The 3-D reconstructions were done under the same threshold for wildtype and Clcn7 -/mice. Cross-sectioned images of the incisors were collected at sequential intervals of 120 μm. In each slice, the mineral density of enamel was measured half-way into the enamel layer within a spot area with a diameter of 6 μm at the mesial, central and lateral side. The mineral density of the enamel, crown dentine, alveolar bone, calvarial and tibial bone was measured at three random sites per section and values averaged per slice. In the incisors, the values for enamel and dentin were plotted against slice number, which represented mineralization with increasing development (from secretory stage to maturation stage). Average values and standard deviation were calculated per mouse and these averages were used to calculate group averages. Since the incisors of the Clcn7 -/mice did not erupt, for the incisors of wild-type controls, enamel values were used from the maturation stage of not yet erupted parts, to rule out post-eruptive changes when teeth are functional and exposed to oral fluids.

Histomorphometric analysis
A point-counting technique (Cruz-Orive and Weibel 1990) was used to measure the bone volume (BV) and total tissue volume (bone volume + bone marrow volume, TV) of the maxillary bone (palatine bone) and mandibular bone (i.e., alveolar bone around molar region representing the alveolar region and condylar bone representing the ascending ramus, separately; Klingenberg et al. 2004).

Statistical analysis
Values are presented as means and standard deviation (SD) and data were tested for statistical differences using Student's t test and one-way ANOVA between the wild-type and Clcn7 -/mice.

Anti-ClC-7 immunostaining of ameloblasts and cells involved in bone remodeling
In jaws of wild-type mice, strong immunostaining with anti-ClC-7 was seen as fine granular material in secretory and maturation ameloblasts (Fig. 1a, d-f). Intense staining was noticed in osteoclasts (Fig. 1e, i) in the apical membrane of maturation ameloblasts and in papillary cells of the enamel organ. Intracellular staining was found in groups of maturation ameloblasts (Fig. 1e, f). Less intense staining was found in odontoblasts (Fig. 1g), bone lining cells (Fig. 1h), osteoblasts (Fig. 1j) and hypertrophic chondrocytes (Fig. 1k). Anti-ClC7 failed to stain enamel organ cells in incisors, developing third molars (Fig. 1b,c) or the giant osteoclasts bordering bone (Fig. 1o) in Clcn7 -/mice. In developing third molars, the enamel organ contained cells with more pronounced apoptotic bodies than in wild-type controls (Fig. 1b, l).

Immunolocalization of Ae2 in developing jaw tissues and long bones of Clcn7 -/mice
To see if disruption of ClC-7 also affected expression of anion exchanger 2 (Ae2), sections were immunostained for Ae2. The basolateral membranes of maturation ameloblasts of Clcn7 -/mice were strongly positive for Ae2 ( Fig. 2a-c) as reported in wild-type mice ). In Clcn7 -/mice, the layer of dental epithelium in the apical part of the incisors had been disrupted and ameloblasts and odontoblasts were trapped by alveolar bone forming odontoma-like structures but the dental epithelium strongly stained for Ae2 (Fig. 2c-e). Huge elongated multinucleated osteoclasts covering the bone surface near the distorted molar roots were also strongly stained for Ae2 (Fig. 2f, g). In both craniofacial bone and tibiae of wild-type mice, the extensive bone marrow cavities contained large numbers of mononuclear cells positive for Ae2. However, no such positive cells were seen in the population of marrow cells in the far less extensive marrow cavities either long bone (Fig. 2i, k) or craniofacial bones in Clcn7 -/mice indicating the composition of the bone marrow cells had changed by disruption of Clcn7 gene. Micro-CT showed substantial differences in mineralized structures of jaw bones, skull and teeth between Clcn7 -/and wildtype mice (Fig. 3). The bone surface of Clcn7 -/mice appeared spongy compared to wild-type controls that had a denser and smoother surface (Fig. 3a, b, g, h). Histology showed that jaw bones in Clcn7 -/mice exhibited a very severe osteopetrotic phenotype as reported for long bones (Fig. 4b) (Kornak et al. 2001). Jaw bones in Clcn7 -/mice contained more trabeculae with many small marrow cavities instead of more mature bone with fewer but larger bone marrow cavities in wild-type controls (Fig. 4b). The trabecular structure of the palatine bone in Clcn7 -/mice was disorganized. Osteocytes in bone of Clcn7 -/mice were larger, round and arranged more irregularly than osteocytes in wild-type mice that were elongated with their long axes parallel to the bone surface. Many of the osteocyte lacunae in bones of Clcn7 -/mice were empty suggesting enhanced apoptosis of osteocytes. The maxillary bone suture was closed in wild-type mice but not in Clcn7 -/mice and retained substantial amounts of cartilage (Fig. 4a, b). The ratio of the width of zone of hypertrophic cartilage to the width of total suture area was 0.34 (±0.03) for wild-type mice and 0.62 (±0.04) for Clcn7 -/mice (n=3, p<0.001).
Electron microscopy revealed extremely long osteoclasts in Clcn7 -/mice containing broad clear zones, many intracellular vesicles and a ruffled border that was poorly developed (Fig. 5a, c). Undecalcified samples of craniofacial bone of the Clcn7 -/mice were easier to section. Collagen bundles of the bone matrix were very loosely packed and between osteoblasts and collagen fibrils of the bone matrix substantial amounts of amorphous nonfibrous material was present (Fig. 5b, e), not seen in wild-type bone (Fig. 5d). The bone surface contained more frequently prominent cementum-like lines to which elongated osteoclasts were attached (Fig. 5b, c). Osteoclasts seemed to resorb some bone and calcified cartilage but far less than controls (Fig. 5a, c). Bone volume of the Clcn7 -/mice was 1.53 fold higher in palatine bone (p<0.001), 1.61-fold higher in alveolar bone (0.01<p<0.05) and 1.43-fold higher in condyle (0.01<p<0.05) than the corresponding parts in wild-type control mice (Table 1). The wild-type incisor is much longer and better shaped than the mutant incisor with a blunt tip. The first molar crown of the mutant mice is well developed but no roots are formed (f). The calvarial bone has also the severe osteopetrotic phenotype such as jaw bones with a smaller dimension and lower mineral density than wild-type control mice (g WT; h KO). Scale bars (a-d, g, h) 1 mm, (e, f) 200 μm Fig. 4 Histological changes of the jaw bones and teeth in Clcn7 -/mice. Palatine bone with suture (S) (a, b). a In wild-type mice (WT), the bone is mature with large bone marrow cavities. b In Clcn7 -/mice, the bone is osteopetrotic with many small marrow cavities. WT incisor with normal wide space between the cervical loop (CL) and bone (B) (c).The radiated arrangement of the normal dentinal tubules of wild-type incisor (c'). The Clcn7 -/mice incisor with wide predentine and folded root (d) and the dentine (D) calcification is abnormal (d'). The molars of Clcn7 -/mice failed to erupt with tissues covering the eruption route (e WT; f KO).

Structural changes in developing teeth
In contrast to wild-type controls, incisors and molars of Clcn7 -/mice failed to erupt into the oral cavity (Figs. 3b,4d). Incisors were often still surrounded by dense bone, incisors were shortened by two-third in comparison with wildtype controls (Fig. 3c, d), but the dental crowns of the molars developed well. However, root formation of the molars was distorted and disfigured by compaction (Fig. 3e, f). In decalcified teeth, no enamel matrix was left in the enamel space and the space was bordered by flattened ameloblasts. Dentine mineralization was irregular, predentine was wider and the dentine tubules were narrower. Parts of incisor dentine were hypomineralized and contained irregular dentin tubules. At the cervical loop of the Clcn7 -/incisors, the dental epithelium layer with the proliferating cells had folded, disrupted and fragments of the layer containing ameloblasts and odontoblasts formed many islands surrounded by bone tissue. Ankylosis occurred locally between dental roots and alveolar bone. Periodontal ligament was poorly developed and the enamel organ occasionally formed cysts (Fig. 4c, d).

Changes in mineral density
In the Clcn7 -/mice, the calcified part of the mandibular incisor was much shorter but the mineral density of enamel still reached values as high as 2493 mg HA per cm 3 , not statistically different from control values that reached maximal values of 2630 mg HA/cm 3 (Fig. 6d-g). Mineral density of incisor dentine was slightly less than the controls (p<0.05).

Discussion
Our data show that ClC-7 is critical for osteoclast functioning in orofacial and calvarial bone as reported for long bones (Kornak et al. 2001;Neutzsky-Wulff et al. 2008). Without functional ClC-7, orofacial bones became severely osteopetrotic and teeth failed to erupt. Impaction of the teeth, root dysplasia and odontoma-like structures were regularly noticed in various osteopetrotic models. So far, these characteristics have been found in five osteopetrotic models, namely ntl -/-, Rank -/-, Rankl -/-, c-src -/and v-H + -ATPase -/mice (Bronckers et al. 2012;Koehne et al. 2013;Lu et al. 2009;Tiffee et al. 1999). Two Clcn7-related osteopetrotic patients suffered from enamel dysplasia and root dysplasia in posterior teeth (Xue et al. 2012). Osteopetrotic cattle with a Clcn7 mutation displayed gross gingival hamartomas, not found in Clcn7 -/mice (Sartelet et al. 2014). We reported here a strong positive immunostaining of wild-type ameloblasts for ClC-7 in line with published data (Lacruz et al. 2013). We also found changes of the dentition (including enamel, dentin, roots and periodontal tissues) in Clcn7 -/mice. We found no evidence that without functional ClC-7 mineralization of enamel was reduced or enamel matrix retained as seen in Cftr -/- (Wright et al. 1996) or Ae2 -/mice (Lyaruu et al. 2008). Basically, the same structural changes in teeth were reported very recently in Clcn7 -/mice in a less extensive way while we prepared this manuscript (Wen et al. 2015). Disruption of Ae2 affects both osteoclasts in long bone and maturation ameloblasts ). Ameloblasts in Clcn7 -/mice stained normally for Ae2 and were not structurally affected. Normal immunostaining for Ae2 was also noticed in basolateral membranes of defective osteoclasts in Clcn7 -/mice, suggesting that osteoclasts expression of Ae2 was not affected and that these osteoclasts are potentially able to import Cl − from the extracellular fluid.
Although teeth failed to erupt, the enamel of Clcn7 -/mice attained the same mineral density as in wild-type enamel and enamel matrix was not retained. These data indicate that ClC-7 is not significantly involved in completion of enamel mineralization as found for ameloblasts deficient for MMP20, KLK4, SPPL2a CFTR, AE2 or NBCe1 (Bartlett et al. 2004;Bronckers et al. 2010Bronckers et al. , 2013Jalali et al. 2014;Lyaruu et al. 2008;Simmer et al. 2009).
In osteoclasts, ClC-7 operates in conjunction with the proton pump associated with the ruffled border, for which the subunit Tcirg1 (T-cell immune regulator 1, also called Atp6v 0 a 3 ) is essential. Both Clcn7 -/mice and v-H + -ATPase null mutation gave an osteopetrotic phenotype but without marked changes in ameloblast function or in enamel mineralization (Bronckers et al. 2012;Wen et al. 2015). Thus, unlike their function in osteoclasts, ClC-7 or v-H + -ATPase are not critical for ameloblast function.
Osteopetrosis is a heterogeneous group of genetic bone disease characterized by an increase in bone volume due to the absence or an impaired activity of osteoclasts (Tolar et al. 2004). Changes in bone formation and in degree of mineralization of osteopetrotic bone, which also influences bone quality (Bollerslev 1989;Tolar et al. 2004;Waguespack et al. 2007), received less attention. In wild-type mice during endochondral ossification, the cartilage is gradually replaced by bone and old bone replaced by new bone (Seeman and Delmas 2006). For Clcn7 -/mice, we found that bone Fig. 6 Mandibular jaw bones of wild-type mice (a) and Clcn7 -/mice (b, c) in a sagittal plane from a 3-D reconstructed micro-CT image. Effect of Clcn7 null mutation on mineral density of developing lower incisors as function of stage (d wild-type mice; e Clcn7 -/mice). 0 on x-axis represents the starting point of maturation stage. Average mineral density of enamel and dentine in maturation stage exclusive the erupted part for wild-type incisors but the whole incisor of Clcn7 -/mice (f) and first molars (g). Average mineral density in alveolar and calvarial bones (h). Scale bars (a-c) 1 mm resorption and bone-turnover were reduced and cartilage was retained. The amount of bone was higher in Clcn7 -/mice but the bone was less mineralized and collagen fibrils less packed and were covered by a layer of amorphous non-collagenous material. This suggested that in vivo mineralization of bone was also impaired. However, in vitro data showed that the ability of osteoblasts of Clcn7 -/mice to mineralize was not altered (Henriksen et al. 2011), suggesting that the changes in bone structure in vivo may be secondary or that in vivo osteoblasts respond differently.
The lower mineral density of bone in Clcn7 -/mice implies that the bone quality and hence the capacity to carry mechanical loads may be reduced in these mice (Del Fattore et al. 2008;Henriksen et al. 2011;Segovia-Silvestre et al. 2009). In young postnatal Clcn7 -/mice, the bone mineral density was lower and the bone contained empty osteocyte lacunae. However, in adult Clcn7 -/mice, bone strength was increased as compared to bone in wild-type mice (Henriksen et al. 2011), suggesting that the changes in bone in early postnatal Clcn7 -/mice could be transient. Apparently, the increased amount of bone mass in Clcn7 -/mice outweighs the reduction in mineral content per unit of bone. However, patients with defective ClC-7 appear to have poor bone quality and suffer from many fractures despite their higher bone mass. This may be related to elevated amounts of calcified cartilage present in bone that weakens the bone structure.
The most commonly affected genes of osteopetrosis are CLCN7 and TCIRG1. Osteopetrosis is frequently accompanied by osteomalacia or osteopetrorickets (Barvencik et al. 2014) and this additional pathology is also found in 2week-old Tcirg1-deficient oc/oc mice. This phenotype was characterized by growth retardation, rachitic widening of the growth plate, marked hyperosteoidosis and diffuse mineralization of the bone surfaces. These defects are the consequence of hypocalcemia resulting from a combined acidification, impairment of osteoclasts and gastric parietal cell. Clcn7 -/mice have some of these characteristics such as growth retardation and widening of the hypertrophic zone in the growth plate. However, hyperosteoidosis was not detected in Clcn7 -/mice nor did these mice display hypocalcemia. Although our micro-CT data showed that the bone mineral density in Clcn7 -/mice was significantly lower than the bones in the wild-type mice, Clcn7 -/mice do not display clear osteopetrorickets at the age of 3 weeks. A novel mutation in CLCN7 (D145 G) impaired the activation and relaxation kinetics of the CLC-7 ion transporter and had no sign of osteomalacia (Barvencik et al. 2014). Thus, in contrast to patients carrying TCIRG1 mutation, patients carrying CLCN7 mutation do not seem to suffer from osteomalacia.
In conclusion, ClC-7 is essential for osteoclasts to resorb craniofacial bones to enable tooth eruption and root development. Disruption of Clcn7 reduces bone and dentin mineral density but does not affect enamel mineralization.


extfieldsofstudy: Biology Medicine

text:
Inhibition of Glutathione Production Induces Macrophage CD36 Expression and Enhances Cellular-oxidized Low Density Lipoprotein (oxLDL) Uptake*

Background: The GSH-dependent antioxidant system reduces atherosclerosis. Results: Inhibition of GSH production by BSO enhanced CD36 translational efficiency to induce CD36 protein expression and lipid accumulation that was blocked by antioxidant (enzyme). Conclusion: Alterations of cellular GSH and GSH/GSSG status regulate macrophage CD36 expression and cellular oxLDL uptake. Significance: Our study demonstrates an important anti-atherogenic function of the GSH-dependent antioxidant system. The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with l-buthionine-S,R-sulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor γ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic function of the GSH-dependent antioxidant system by providing a novel molecular mechanism.

The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with L-buthionine-S,Rsulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor ␥ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic func-tion of the GSH-dependent antioxidant system by providing a novel molecular mechanism.
Formation of lipid-laden macrophage/foam cells is the initial and critical step in the development of atherosclerosis, which is a major cause of coronary heart disease. Disruption of glutathione (GSH) biosynthesis, metabolism, and functions has been implicated in the development of atherosclerosis (1)(2)(3)(4). The decreased cellular GSH levels can result in overproduction of cellular reactive oxygen species (ROS), 3 particularly by macrophages, which can cause multiple biological alterations such as increased production of inflammatory molecules and oxidatively modified products like the oxidized low-density lipoprotein (oxLDL). Uptake and internalization of oxLDL by macrophages can enhance cellular lipid accumulation and foam cell formation (5).
CD36 has been defined as a receptor for oxLDL and plays an important role in foam cell formation and development of atherosclerosis (6,7). The uptake of oxLDL by human monocytederived macrophages is blocked by anti-CD36 antibody by 50% (8). Monocytes isolated from humans lacking functional CD36 expression exhibit significantly decreased capacity for oxLDL binding (9). In animal models, genetic deletion of CD36 expression reduces foam cell formation and inhibits the development of atherosclerosis in apoE deficient (apoE Ϫ/Ϫ ) mice (10).
We previously reported that oxLDL induced macrophage CD36 expression, which suggests a positive feedback loop driving foam cell formation by oxLDL (11). Studies on the mechanism(s) responsible for oxLDL-induced CD36 expression have discovered the critical role of peroxisome proliferator-activated receptor ␥ (PPAR␥), a ligand-activated transcription factor, in regulating CD36 transcription (12,13). Activation of PPAR␥ by ligand results in formation of a heterodimer of PPAR␥ with another transcription factor, retinoid X receptor ␣ (RXR␣). The complex of PPAR␥/RXR␣ binds to the PPAR␥-responsive element in the proximal region of the promoter in target genes including CD36 to initiate their transcription (14). Two of the major oxidized lipid components in oxLDL, 9-hydroxyoctadecadienoic acid (9-HODE) and 13-HODE, have been identified as the endogenous ligands for PPAR␥ (13), suggesting that oxLDL induces macrophage CD36 expression and foam cell formation in a PPAR␥-dependent manner. CD36 expression can also be regulated by cellular glucose levels by a translational mechanism (15).
GSH (glutamate-cysteine-glycine) is a tripeptide thiol and is produced by most mammalian cells at up to millimolar concentrations. Most cellular GSH (80 -85%) is present in cytoplasm because of its hydrophilic property (16). In addition to being a co-factor for various enzymatic reactions, GSH can function as an antioxidant to reduce cellular ROS levels. The GSH-dependent antioxidant system has been demonstrated to play an important role in regulating foam cell formation and development of atherosclerosis. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in regulating cellular GSH production (17). Genetic deletion of GCL expression enhances atherosclerosis, whereas high expressing GCL inhibits atherosclerosis in apoE deficient (apoE Ϫ/Ϫ ) mice (1). Glutathione peroxidase-1 (GPx-1) is an antioxidant enzyme that uses GSH as the substrate to convert hydrogen peroxide or lipid peroxides into water or the respective alcohols. Deficiency of GPx-1 expression accelerates progression of atherosclerosis (2,18). Glutathione reductase maintains cellular GSH homeostasis by catalyzing reduction of glutathione disulfide (GSSG) into GSH using NADPH as the reducing cofactor. Increased macrophage glutathione reductase expression also inhibits atherosclerosis in LDL receptor-deficient (LDLR Ϫ/Ϫ ) mice (3).
Despite the above findings, the underlying mechanisms responsible for the anti-atherogenic properties of the GSH-dependent antioxidant system have not been fully elucidated. Because of the importance of CD36 in macrophage oxLDL uptake and foam cell formation, in this study we investigated if inhibition of macrophage GSH production by a potent specific GCL inhibitor, L-buthionine-S,R-sulfoximine (BSO) (19), or regulation of cellular GSH/GSSG status can directly mediate macrophage CD36 expression and cellular oxLDL uptake. We also attempted to determine the involved mechanisms.
Cell Culture-RAW264.7 cells, a murine macrophage cell line, were purchased from ATCC (Rockville, MD) and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 50 g/ml penicillin and streptomycin. Peritoneal macrophages were collected from mice as described (20), and purity was determined by immunofluorescent staining with anti-MOMA-2 antibody. Cells received treatment in serum-free medium.
In Vivo Study-The protocol for animal study was approved by the Animal Ethics Committee of Nankai University and conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. C57BL/6 wild type and apoE Ϫ/Ϫ mice were purchased from the Animal Center of Nanjing University (Nanjing, China). To generate macrophage PPAR␥-deficient mice, the homozygous floxed (ϩ/ϩ) PPAR␥ mice (kindly provide by Dr. S. Duan) crossbred with the transgenic mice containing the CRE gene under the control of the murine M lysozyme promoter (CRE-M lysozyme (ϩ/ϩ)) mice (21). Therefore, the control mice are flox ϩ/ϩ /CRE Ϫ/Ϫ , and the conditional macrophage PPAR␥ knock-out mice are flox ϩ/ϩ /CRE ϩ/Ϫ . The peritoneal macrophages isolated from these two types of mice are defined as PPAR␥ fl/fl and MacPPAR␥ KO cells, respectively.
To determine the effect of decreased GSH on CD36 expression in vivo, apoE Ϫ/Ϫ mice were fed normal chow or normal chow plus BSO (50 mg/100 g of food) for 1 week. Based on the food consumption, the dose of BSO was estimated to be ϳ0.25 mmol/kg (bodyweight)/day. At the end of treatment, all of the mice were anesthetized and euthanized in a CO 2 chamber followed by collection of peritoneal macrophages and kidneys. The cells were lysed while a piece of kidney was homogenized in a lysis buffer (20 mM Tris, pH 7.5, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM ␤-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin/leupeptin, 100 mM NaVO 4 ). The cellular lysate or kidney homogenate was centrifuged for 10 min at 16,000 ϫ g at 4°C. The supernatant was transferred into a new test tube as total protein extract for determination of protein expression by Western blot.
Determination of Cellular GSH and GSSG Levels-Cellular GSH and GSSG levels were determined by enzymatic recycling method (22) using assay kits. The cell samples for GSH/GSSG assay were prepared as follows; after treatment and washing with cold Ca 2ϩ /Mg 2ϩ -free PBS, macrophages were suspended in ice-cold extraction buffer (1% Triton X-100 and 0.6% sulfosalicylic acid in 0.1 M K 3 PO 4 , 5 mM EDTA, pH 7.5) and homogenized. A portion of the homogenate was saved for determination of the cellular protein content, which was used to normalize cellular GSH and GSSG levels. The rest of the homogenate was centrifuged for 5 min at 10,000 ϫ g at 4°C, and the supernatants were used for GSH/GSSG assay using the assay kit based on the manufacturer's instructions. To prepare kidney samples for GSH determination, a piece of kidney was weighed and homogenized in an ice-cold mixture of 5% met-aphosphoric acid and 0.6% sulfosalicylic acid. The supernatant of homogenate was used to determine GSH content, which was normalized by the weight of the piece of kidney and expressed as mol/g of tissue.
Determination of Cellular ROS Levels-The intracellular ROS levels were determined using a ROS assay kit purchased from Beyotime (Nantong, China) that is based on the principle that oxidation of the non-fluorescent probe 2Ј,7Ј-dichlorodihydrofluorescein diacetate by intracellular ROS can generate the highly fluorescent 2Ј,7Ј-dichlorofluorescein (23). Briefly, after treatment, 5 M 2Ј,7Ј-dichlorodihydrofluorescein diacetate solution was added to the cells in a 96-well plate and incubated in the dark for 20 min at room temperature followed by washing with PBS 3 times. The 2Ј,7Ј-dichlorofluorescein fluorescence generated within the cells was determined at 485-nm (excitation) and 527-nm (emission) wavelengths on a fluorescence microplate reader (EnSpire, PerkinElmer Life Sciences). After determination of fluorescence, the cells remaining in each well of the plate were lysed, and cellular protein content was determined that is used to normalize the 2Ј,7Ј-dichlorofluorescein fluorescence. The normalized 2Ј,7Ј-dichlorofluorescein fluorescence intensity of control samples was defined as 100%.
Determination of Macrophage oxLDL Uptake-After treatment, macrophages were incubated with rabbit anti-CD36 antibody or normal IgG for 1 h followed by incubation with oxLDL (50 g/ml) for 3 h. The cells were then fixed in 4% paraformaldehyde for 30 min at room temperature, washed twice with PBS for 5 min, and stained with Oil Red O solution (a mixture of three parts 0.5% Oil Red O in isopropyl alcohol and 2 parts water) for 50 min at room temperature followed by washing twice with water. The cells were counterstained with hematoxylin solution for nuclei for 30 s, kept in water for 5 min, and then photographed. The accumulated Oil Red O dye within macrophages was extracted by added isopropyl alcohol, and the absorbance of the extraction solution was determined at 510 nm. After normalized by cellular protein content, the absorbance of the control group was defined as 1.
Isolation of Total RNA and Determination CD36 mRNA Expression-Total cellular RNA was extracted as described (20). The cDNA was synthesized with 1 g of total cellular RNA using the reverse transcription kit purchased from New England BioLabs (Ipswich, MA). PCR was performed with the following primers: CD36 forward, (5Ј-TTTCCTCTGACATTTG-CAGGTCTA-3Ј) and reverse, (5Ј-AAAGGCATTGGCTGGA-AGA-3Ј) and GAPDH forward (5Ј-ACCCAGAAGACTGTG-GATGG-3Ј) and reverse, (5Ј-ACACATTGGGGGTAGGAACA-3Ј). The RT-PCR product was determined by agarose electrophoresis and photographed. The real time PCR was performed with the above primers and an SYBR Green PCR master mix purchased from Bio-Rad. Expression of CD36 mRNA was normalized by the corresponding GAPDH mRNA.
Western Blot Analysis of CD36, STAT1, and Phosphorylated STAT1 (pSTAT1), FACS, and Immunofluorescent Staining Analysis of CD36 -After extraction, an equal amount of cellular protein from each sample was used to determine CD36, STAT1, and pSTAT1 protein levels by Western blot as described (20).
To analyze cell surface CD36 protein levels, ϳ1 ϫ 10 6 cells from each sample were blocked for 30 min at room temperature with PBS containing 5% goat serum. After washing, the cells were incubated with anti-CD36 antibody (1:100) for 1 h at room temperature followed by incubation with goat anti-rabbit FITC-conjugated IgG (1:50) for 45 min and flow cytometric evaluation.
CD36 expression was also determined by immunofluorescent staining as described (20). Images of cells were obtained with a fluorescence microscope (Leica). The mean fluorescence intensity of all the immunofluorescent images was determined as described (24).
Determination of CD36 Protein Translational Efficiency-CD36 protein translational efficiency was determined by two methods: polysomal RNA fractioned-Northern blot analysis and surface sensing of translation (SUnSET) technique. The polysomal RNA fractioned-Northern blot analysis was completed as follows (15,25). After treatment and washing with PBS, ϳ10 8 cells were resuspended in 0.75 ml of a low salt buffer (20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2 ) containing Triton X-100 (1.2%, v/v) and sucrose (0.2 M) and homogenized. After centrifugation for 30 s at 27,000 ϫ g at 4°C, the supernatant was mixed with 0.1 ml of low salt buffer containing heparin (10 mg/ml). After the addition of NaCl (final 0.15 M), the mixture was applied to the top of a 0.5-1.5 M linear sucrose gradient (9.6 ml in low salt buffer) and centrifuged for 3 h at 32,000 ϫ g at 4°C. The gradients were fractionated into 6 fractions (1 ml/fraction). SDS and proteinase K (final concentration: 0.5% and 0.1 mg/ml, respectively) were added to each fraction and mixed well. The mixture was then incubated for 30 min at 37°C followed by RNA extraction. Expression of CD36 mRNA in the polysomal RNA fractions was determined by Northern blot (26).
For SUnSET the technique assay (27)(28)(29), macrophages were treated with BSO for 12 h followed by treatment with puromycin (10 g/ml) for 1 h. After whole cellular protein extraction, the input was determined by Western blot with anti-puromycin antibody. Based on the results of input, the same amount of cellular protein from each sample was used to conduct immunoprecipitation by anti-CD36 antibody or normal IgG followed by Western blot analysis with anti-puromycin, anti-CD36, or anti-IgG antibody.
Preparation of Plasmid DNA and Determination of CD36 Promoter Activity-Mouse CD36 promoter (from Ϫ1946 to ϩ51) was constructed by PCR with genomic DNA isolated from RAW264.7 cells and the following primers: forward (5Ј-CCG-CTCGAGAAGTGCAGAAGTTTACATGGG-3Ј) and reverse (5Ј-CCCAAGCTTAGGAGCTGTCTTCCAGGTG-3Ј). After the sequence was confirmed, the PCR product was digested with XhoI and HindIII followed by ligation with pGL4 luciferase reporter vector, transformed into Escherichia coli to amplify.
To determine CD36 promoter activity, ϳ95% confluent 293T cells were transfected with DNA for CD36 promoter, PPAR␥, and RXR␣ expression vectors and Renilla (for internal normalization) using Lipofectamine TM 2000 (Invitrogen). After 20 h of transfection plus treatment, the cells were lysed, and cellular lysate was used to determine firefly and Renilla

Cellular GSH/GSSG Status Regulates CD36 Expression
luciferase activity using the Dual-Luciferase Reporter Assay System from Promega (Madison, WI).
Macrophages were transfected with scrambled siRNA, GCL siRNA, or CD36 siRNA with the transfection reagent purchased from Origene (Rockville, MD) in an antibiotics/serumfree RPMI 1640 medium for 24 h. The cells were then switched into complete medium and continued culture or culture or plus treatment for another 48 h. The cells transfected with GCL siRNA were used to extract cellular total RNA and protein followed by determination of GCL mRNA expression by RT-PCR with the forward and reverse primers (5Ј-CTGCACATCTAC-CACGCAGT-3Ј and 5Ј-GTCTCAAGAACATCGCCTCC-3Ј, respectively) and normalized by GAPDH mRNA, CD36 protein expression by Western blot, and cellular GSH levels by assay kit. The cells transfected with CD36 siRNA were treated with BSO or BSO plus antioxidant (enzyme) overnight followed by determination of oxLDL uptake/foam cell formation by incubating cells with oxLDL and ROS production by the ROS assay kit, as described above.
Data Analysis-All experiments were conducted at least three times, and representative results are presented. Data were analyzed by Student's t test (n ϭ 3), and significant difference was considered at p Ͻ 0.05.

Cellular GSH/GSSG Status Regulates Macrophage CD36
Protein Expression-Reaction of GSH with ROS generates GSSG, which can be reduced back to GSH by NADPH. Both cellular GSH content and the ratio of GSH to GSSG (GSH/ GSSG) play an important role in anti-oxidative stress. GSH is mainly synthesized by kidney in vivo because the highest GCL activity is in this tissue (30). However, other tissues/cell types can also produce GSH. To study if the alterations of cellular GSH levels or GSH/GSSG ratios can influence macrophage CD36 expression, RAW264.7 macrophages were treated with BSO. BSO reduced cellular GSH levels in a concentration-dependent manner (Fig. 1A, left panel). Associated with decreased GSH levels, cellular GSH/GSSG ratios were also reduced ( Fig.  1A, right panel), whereas cellular ROS levels were increased (Fig. 1B). The results of a Western blot show that BSO increased CD36 protein expression in a concentration-dependent manner (Fig. 1C, left panel; normally, two bands and one band of CD36 protein were determined in RAW264.7 cells and peritoneal macrophages or kidney, respectively). The time course study indicates that BSO induced CD36 protein expression in a time-dependent fashion (Fig. 1C, right panel).
CD36 is a membrane protein.
To determine the effect of BSO on cell surface CD36 protein levels, after treatment cells were subjected to flow cytometric evaluation. Fig. 1D demonstrates that BSO increased cell surface CD36 protein lev-els. The effect of BSO on CD36 protein expression in primary cells was determined by immunofluorescent staining. Similar to RAW264.7 cells, BSO also induced peritoneal macrophage CD36 protein expression in a concentration dependent manner (Fig. 1, E and F).
To directly link cellular GSH production to macrophage CD36 expression, we selectively inhibited GCL expression by siRNA. Compared with scrambled siRNA, GCL siRNA inhibited GCL mRNA expression (Fig. 1G). Associated with inhibition of GCL expression, cellular GSH levels were decreased (Fig. 1H), whereas expression of macrophage CD36 protein expression was induced (Fig. 1I). Taken together, the results in Fig. 1 suggest that inhibition of cellular GSH production can induce macrophage CD36 protein expression.
Induction of CD36 protein expression by BSO can result in increased cellular oxLDL uptake, which facilitates foam cell formation. To determine it, after BSO treatment macrophages were conducted in Oil Red O staining. The top panel of Fig. 2A shows that BSO treatment led to increased oxLDL uptake, indicating formation of foam cells. The quantitative analysis of accumulated Oil Red O dye within cells (Fig. 2B) also demonstrates that the increased cellular lipid accumulation is in a concentration-dependent manner. However, blocking CD36 function by anti-CD36 antibody substantially attenuated lipid accumulation ( Fig. 2A, bottom panel), suggesting that the enhanced macrophage oxLDL uptake and foam cell formation by BSO is mainly completed through induction of CD36 expression, which was also confirmed by a study with CD36 siRNA (Fig. 3F).
The decreased cellular GSH levels and GSH/GSSG ratios by BSO increase cellular ROS production (Fig. 1B), which can enhance production of oxLDL if LDL is present (31). Consequently, the generated oxLDL can activate CD36 expression. To test it, macrophages were treated with LDL or LDL plus BSO. LDL alone had little effect on CD36 expression. However, co-treatment of macrophages with BSO and LDL further enhanced BSO-induced CD36 expression (Fig. 2C), which should be attributed to the oxidation of LDL in response to BSO treatment. In fact, the results of an oxLDL ELISA assay demonstrate that BSO substantially increased cellular oxLDL content in the presence of LDL (Fig. 2D).
We next determined if amelioration of cellular GSH/GSSG status by exogenous GSH or other antioxidant or antioxidant enzyme can attenuate BSO-induced macrophage CD36 protein expression. Fig. 3A demonstrates that treatment of macrophages with exogenous GSH decreased CD36 expression. In the presence of a small molecular antioxidant, such as N-acetyl cysteine (NAC), and antioxidant enzyme (catalase (CAT) or superoxide dismutase (SOD)), the decreased GSH/GSSG ratio was restored to normal (Fig. 3B). Macrophages greatly express inducible nitric-oxide synthase to produce a large amount of nitric oxide when cells receive stimuli. Nitric oxide can also induce cellular oxidative stress. However, treatment of macrophages with N G -monomethyl-L-arginine (L-NMMA, a potent inducible nitric-oxide synthase inhibitor) did not affect the BSO-decreased GSH/GSSG ratio because BSO inhibited inducible nitric-oxide synthase expression (32). Similarly, CAT or NAC or SOD alone slightly inhibited or had no effect on CD36 SEPTEMBER 4, 2015 • VOLUME 290 • NUMBER 36

JOURNAL OF BIOLOGICAL CHEMISTRY 21791
expression, but each of them blocked CD36 protein expression induced by BSO. Meanwhile, L-NMMA had no effect on CD36 expression in either the absence or presence of BSO (Fig. 3C). Furthermore, amelioration of cellular GSH/GSSG status by CAT, SOD, and NAC attenuated BSO-induced cellular lipid accumulation (Fig. 3D, left half). However, anti-CD36 antibody abolished the effect of BSO or BSO plus antioxidant (enzyme) on oxLDL uptake (Fig. 3D, right half), which suggests that the regulation of cellular oxLDL uptake by BSO or BSO plus antioxidant (enzyme) is contributed by CD36 expression. Correspondingly, BSO increased ROS production, which was blocked by CAT, SOD, and NAC but not NMMA (Fig. 3E).
To further confirm that the BSO-induced cellular oxLDL uptake is mainly contributed by induction of CD36 expression, we transfected macrophages with CD36 siRNA to selectively inhibit CD36 expression (Fig. 3F, right panel) and then determined effect of BSO or BSO plus antioxidant (enzyme) on oxLDL uptake. Similar to normal macrophages (Fig. 3D), BSO treatment substantially induced lipid accumulation in cells transfected with scrambled siRNA (Ctrl siRNA), whereas CAT, SOD, and NAC, but not NMMA, blocked BSO-induced cellular oxLDL uptake (Fig. 3F, left panel). In contrast, cells lacking CD36 expression by CD36 siRNA demonstrate reduced oxLDL uptake at the basal levels, whereas BSO or BSO plus antioxidant (enzyme) had little effect on it (Fig. 3F, middle panel) which further suggest that BSO induced cellular oxLDL uptake is completed through induction of CD36 expression.

Induction of CD36 Expression by GSH Depletion in Vivo-
To determine if the decreased GSH levels can induce CD36 expression in vivo, particularly in pro-atherogenic apoE Ϫ/Ϫ mice, apoE Ϫ/Ϫ mice were fed normal chow or normal chow plus BSO (50 mg/100 g of food or ϳ0.25 mmol/kg (bodyweight)/day) for 1 week. Compared with control mice (chow alone), no difference in food intake, bodyweight gain, and exterior appearance was caused by BSO treatment. Among mouse tissues, the highest GCL expression has been reported in the kidney (30). Therefore, both mouse peritoneal macrophages and kidneys were collected to determine GSH levels and CD36 expression. BSO decreased mouse kidney and peritoneal macrophage GSH levels to ϳ15 and ϳ25% of controls (Fig. 4, bottom of each image), respectively. Associated with decreased GSH levels, expression of CD36 in both peritoneal macrophages and kidneys was increased (Fig. 4).
BSO Induces CD36 Protein Expression by Enhancing CD36 Translational Efficiency-To determine if BSO induces CD36 expression at a transcriptional level, we initially determined macrophage CD36 mRNA expression in response to BSO treatment. The results in Fig. 5A show that BSO had little effect on CD36 mRNA levels, which implies that the induction of CD36 protein expression does not occur at the transcriptional level. Furthermore, we determined that antioxidant or antioxidant enzyme alone or in combination with BSO had little effect on CD36 mRNA expression either (Fig. 4B), which indicates that antioxidant (enzyme) blocks BSO-induced CD36 protein expression at a post-transcriptional level also. In contrast to BSO alone, co-treatment of cells with LDL and BSO increased CD36 mRNA expression (Fig. 5C) because the co-treatment generated oxLDL (31) (Fig. 2D), which activates CD36 transcription (11,13).
To determine the effect of BSO on CD36 mRNA stability, macrophages were treated with actinomycin D or actinomycin D plus BSO for different times followed by determination of CD36 and GAPDH mRNA levels. Actinomycin D can potently inhibit cellular RNA transcription. Thus, mRNA levels in the presence of actinomycin D demonstrate the rate of mRNA degradation and mRNA stability indirectly. Fig. 5D shows similar CD36 mRNA levels between the group receiving actinomycin D alone treatment and the group receiving actinomycin D plus BSO treatment, suggesting that BSO has no effect on CD36 mRNA stability.
CD36 transcription is mainly controlled by transcription factor of PPAR␥. To further confirm that BSO has no effect on CD36 transcription, we constructed a CD36 promoter (from  Ϫ1946 to ϩ51), which includes the PPAR␥-responsive element motif. As shown in Fig. 5E, high expression of PPAR␥ and RXR␣ induced CD36 promoter activity, and the induction was further enhanced by a PPAR␥ ligand, pioglitazone. However, BSO had little effect on CD36 promoter activity either in the absence or presence of pioglitazone (Fig. 5E).
To further confirm that the induction of CD36 protein expression by BSO is independent of PPAR␥ activity, we iso-  (n ϭ 3). E, the cells in Fig. 3D were also determined ROS production by assay kit. *, p Ͻ 0.05 (n ϭ 3). DCF, dichlorofluorescein. F, peritoneal macrophages were transfected with scrambled siRNA (Ctrl siRNA) or CD36 siRNA (50 nM of each) and then treated with CAT, SOD, NAC, and NMMA alone or each plus BSO. Inhibition of CD36 expression was determined by real time RT-PCR (right panel). **, p Ͻ 0.01 (n ϭ 3). Cellular oxLDL uptake and foam cell formation were determined by incubation with oxLDL followed by Oil Red O staining (left and middle panels) and quantitation of accumulated Oil Red O dye within cells. *, p Ͻ 0.05 (n ϭ 3). lated peritoneal macrophages from the conditional macrophage PPAR␥ knock-out mice and the corresponding control mice and then treated the cells with BSO. As expected, deficiency of PPAR␥ expression reduced CD36 expression at the basal levels (Fig. 5F), which suggests the importance of PPAR␥ in CD36 expression. Treatment of control cells with BSO did not affect PPAR␥ expression. Furthermore, BSO induced CD36 protein expression in both control macrophages and PPAR␥deficient macrophages (Fig. 5F, left panel) at a similar degree (Fig. 5F, right panel), indicating that PPAR␥ is not involved in BSO-induced CD36 protein expression.
15(S)-Hydroxyeicosatetraenoic acid, the major 15-lipoxygenase 1/2 metabolite of arachidonic acid, has been reported to induce macrophage CD36 expression and foam cell formation through xanthine oxidase and NADPH oxidase-dependent ROS production and STAT1 activation at both transcriptional and translational levels (33). To exclude the induction of CD36 expression by BSO is related to STAT1 activation, we determined expression of total STAT1 (STAT1) and pSTAT1 in response to BSO treatment. The results in Fig. 5G, left panel, demonstrate that BSO has little effect on both STAT1 and pSTAT1 levels. In addition, the presence of antioxidant enzyme, such as CAT, had no effect on STAT1 and pSTAT1 levels either (Fig. 5G, right panel), which suggests that STAT1 activation is not involved in BSO-induced CD36 expression. Taken together, the results in Fig. 5 demonstrate that BSO does not affect CD36 transcription, and neither PPAR␥ nor STAT1 is involved in BSO induced CD36 protein expression. It also implies that the induction of CD36 protein expression by BSO should occur at the translational level.
To determine the mechanisms by which BSO induced CD36 protein expression, we initially determined if BSO can increase CD36 protein stability. Cycloheximide is a potent inhibitor of cellular protein biosynthesis. Similar to actinomycin D, CD36 protein levels in the presence of cycloheximide demonstrate the rate of CD36 protein degradation and CD36 protein stability indirectly in response BSO treatment. As shown in Fig. 6A, the similar reduction of CD36 protein levels was determined in the presence of cycloheximide alone and cycloheximide plus BSO, which suggests that BSO had no effect on CD36 protein stability.
The above results imply that induction of CD36 protein expression by BSO should occur through enhanced CD36 translation. To determine it, we performed the following two experiments. We initially conducted the polysomal RNA fractioned-Northern blot analysis. After BSO treatment, polysomal fractions were prepared from cells by a sucrose gradient centrifugation, and RNA extracted from each fraction was then subjected to Northern blot analysis. The results of Northern blot demonstrate that distribution of GAPDH mRNA in each of the sucrose gradient fractions was similar between control and BSO-treated macrophages. In contrast, more CD36 mRNA derived from BSO-treated macrophages than control cells was found in the heaviest fractions (Fig. 6B, fractions 5 and 6), indicating that BSO increases the number of ribosomes/transcript and enhances CD36 translational efficiency.
The effect of BSO on CD36 protein translation was further determined by SUnSET analysis (27)(28)(29). Puromycin can effectively end label newly synthesized proteins (peptides), which can be detected by Western blot with anti-puromycin antibody. Specifically, after immunoprecipitation by anti-CD36 antibody, the newly synthesized CD36 protein can be determined by Western blot with anti-puromycin antibody. Fig. 6C, left panel, demonstrates that BSO had little effect on the global protein translation. However, compared with control sample (Fig. 6C,  lane 2, right panel), after immunoprecipitated by anti-CD36 antibody, more puromycin-labeled CD36 protein was determined in samples with BSO treatment (Fig. 6C, lanes 3 and 4,  right panel) by Western blot with anti-puromycin antibody. Therefore, by using two different assays, we demonstrate that BSO can enhance CD36 protein translational efficiency.

Discussion
Although antioxidant vitamins, such as vitamins C and E, can reduce ROS production and suppress oxidative stress, the role played by these vitamins in atherosclerosis is still controversial. Recent large clinical trials do not show the favorable effects of antioxidant vitamins on the established atherosclerosis (34,35). In animal models, selective macrophage ascorbic acid (vitamin C) deficiency surprisingly suppresses early atherosclerosis (36). In contrast, combined vitamin C and E deficiency enhances the development of atherosclerosis in apoE Ϫ/Ϫ mice, in particular the mice also lacking vitamin C biosynthesis by genetic deleting gulonolactone oxidase (gulo) expression (37). Vitamin E, in combination with coenzyme Q10 (CoQ 10 ) or selenium, also reduces atherosclerosis in apoE Ϫ/Ϫ mice or rabbits (38,39).
GSH is an abundant endogenous antioxidant and protects cells against the oxidative stress-induced cell injuries. The antiatherogenic properties of the GSH-dependent antioxidant system have been determined at different levels. Clinically, an inverse correlation between the activity of red blood cell GPx-1 and the risk of cardiovascular events in patients has been reported (4). Polymorphisms in the 5Ј flanking region of the GCL catalytic subunit (GCLC) and GCL modifier subunit (GCLM) genes in patients results in decreased plasma GSH levels and increased risk of myocardial infarction and endothe- lial dysfunction (40,41). In animal models alterations of expression of molecules involved in GSH production, utilization, and cycles, such as GCL, GPx-1, and glutathione reductase, influence atherosclerosis in both LDLR Ϫ/Ϫ and apoE Ϫ/Ϫ mice (3,18). Several actions can make contributions to the anti-atherogenic properties of GSH-dependent antioxidant system. For instance, GCL protects macrophages from mitochondrial hyperpolarization induced by oxLDL (3). Deficiency of GPx-1 results in activation of pro-inflammatory and pro-fibrotic pathways (2). Administration of LDLR Ϫ/Ϫ mice with streptozotocin decreases GSH/GSSG ratios in macrophages and enhances macrophage recruitment into aortic wall, thereby accelerating the development of atherosclerosis (42).
The formation of foam cells is the initial and critical step in the development of atherosclerosis, and it is completed by uptake of modified LDL (mainly oxLDL) by macrophage scav-  (n ϭ 3). D, RAW264.7 cells were treated with actinomycin D (ActD, 5 g/ml) or actinomycin D plus BSO (5 M) for the indicated times. Expression of CD36 and GAPDH mRNA was determined by RT-PCR. E, ϳ90% confluent 293T cells were transfected with DNA for the CD36 promoter, PPAR␥ and RXR␣ expression vectors, and Renilla luciferase for 4 h followed by treatment with BSO or BSO plus pioglitazone overnight. After transfection and treatment, the cellular lysate was extracted for determination of the activity of firefly and Renilla luciferases. F, peritoneal macrophages isolated from the PPAR␥ fl/fl and the conditional macrophage PPAR␥ knock-out (MacPPAR␥ KO) mice were treated with BSO at the indicated concentrations overnight followed by determination of PPAR␥ and CD36 protein expression. G, RAW264.7 cells were treated with BSO at the indicated concentrations (left panel) or BSO plus CAT as indicated (right panel) overnight. Expression of total STAT1 and pSTAT1 was determined by Western blot. enger receptors including CD36. Formation of foam cells is also influenced by the balance between pro-oxidants and antioxidants. Antioxidants can prevent LDL oxidation as well as cellular lipid accumulation (43). In contrast, the overproduction of ROS can enhance oxLDL generation. It may also result in activation of molecules, which are responsible for oxLDL uptake. For instance, activation of Toll-like receptor 9 by CpG oligodeoxynucleotide increases ROS production and activates expression of lectin-like oxLDL receptor-1 (Lox1) at the transcriptional level, another macrophage scavenger receptor for oxLDL, thereby enhancing oxLDL uptake and formation of foam cells (44).
CD36 facilitates foam cell formation by binding and internalizing oxLDL. Expression of macrophage CD36 can also be regulated by cellular oxidative status. Treatment of macrophages with 15(S)-hydroxyeicosatetraenoic acid enhances ROS production, which results in increased oxLDL uptake and foam cell formation (33). In contrast, vitamin E or ␣-tocopherol decrease macrophage CD36 expression and oxLDL uptake (45,46). Similarly, in our study treatment of macrophage with BSO decreased cellular GSH levels and GSH/GSSG ratios while increasing ROS production, CD36 expression, and cellular oxLDL uptake (Figs. 1 and 2). In contrast, amelioration of cellular GSH/GSSG status by antioxidant (NAC) and antioxidant enzymes (CAT and SOD) restored BSO-decreased GSH/GSSG ratios thereby blocking BSO-induced CD36 expression and oxLDL uptake (Fig. 3, B-D). Induction of CD36 expression by GCL siRNA (Fig. 1, G and I) indicates the direct effect of cellular GSH/GSSG status on cellular oxLDL uptake. Lack of CD36 expression abolished BSO-induced oxLDL uptake (Fig. 3F), demonstrating the direct contribution by BSO-induced CD36 expression.
Expression of CD36 is transcriptionally activated by PPAR␥. Regulation of CD36 expression by vitamin E and 15(S)-hydroxyeicosatetraenoic acid is also completed at the transcriptional level (33,45,46). In contrast, in our study, although we determined that BSO substantially induced macrophage CD36 protein expression and cellular oxLDL uptake in a CD36-dependent manner while the induction was blocked by antioxidant (enzyme), we observed that BSO or BSO plus antioxidant (enzyme) had little effect on CD36 mRNA levels and promoter activity (Fig. 5, A-D). In addition, we excluded the involvement of PPAR␥ and STAT1 in BSO-induced CD36 expression (Fig. 5, E-G). These results suggest that the post-transcriptional regulation should play a critical role in BSO-induced CD36 expression. In fact, we demonstrated that BSO enhanced CD36 translational efficiency (Fig. 6).
The translational regulation of CD36 expression in different cell types has been reported by a few studies. In macrophages, glucose increased CD36 translation due to the existence of the specific features of CD36 transcript. A 5Ј-UTR length of Ͼ200 bp that contains 3 upstream open reading frames (uORFs) and several stable predicted secondary structures exists in CD36 transcript. The first ORF plays a critical role for induction of CD36 translation in response to glucose treatment by increasing translation reinitiation (15). In hepatocytes, activation of mTOR signaling pathway is involved in regulation of CD36 translation. Induction of inflammatory stress in HepG2 cells by TNF-␣ and IL-6 treatment or in mice by administration of casein increases cellular lipid accumulation and CD36 protein expression through enhancement of CD36 translational efficiency, which is blocked by rapamycin, an mTOR-specific inhibitor (47). The induction of hepatic CD36 translation by palmitate is also completed through activation of the mTOR The cells were then used to prepare polysomal RNA fractions followed by determination of CD36 and GAPDH mRNA expression by Northern blot analysis. C, RAW264.7 cells were treated with BSO at the indicated concentrations for 12 h followed by treatment with puromycin (10 g/ml) for 1 h. After treatment, the cellular protein was extracted and used to conduct input assay by Western blot (IB) with anti-puromycin antibody (left panel). After input assay, the protein samples were used to conduct immunoprecipitation (IP) with anti-CD36 antibody or normal IgG followed by Western blot with anti-puromycin antibody for newly synthesized CD36 protein or with anti-CD36 antibody for whole CD36 protein expression (right panel).
signaling pathway (48). However, the interaction of ROS and mTOR is still unclear. Cadmium induces ROS production to activate mTOR pathway, leading to neuronal cell death (49). In contrast, mTOR pathway is inhibited by hydrogen peroxide (49). Salvianolic acid A activates mTOR by reducing ROS production (50). In our study inhibition of GSH production by BSO or GCL siRNA induced macrophage CD36 expression and enhanced oxLDL uptake, which was associated with increased ROS production and blocked by antioxidant (enzyme). Although we determined the induction of CD36 expression occurred at the translational level, it needs more investigation to unveil if this induction is related to enhanced reinitiation of CD36 translation or activation of mTOR signaling pathway.
In summary, our study not only demonstrates that macrophage CD36 expression and oxLDL uptake can be regulated by multiple mechanisms including the translational regulation but also suggests an anti-atherogenic property of GSH-dependent antioxidant system by providing cellular and molecular evidence.


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Characterization of 12 polymorphic SSR markers in Veronica subsect. Pentasepalae (Plantaginaceae) and cross-amplification in 10 other subgenera1

Premise of the study: Microsatellite primers were developed in the perennial herbs of the diploid-polyploid complex Veronica subsect. Pentasepalae (Plantaginaceae) to investigate the role that hybridization has played in the evolution of the group, which includes several endangered species. Methods and Results: Twelve pairs of primers leading to polymorphic and readable markers were identified and optimized from V. jacquinii and V. orbiculata using a microsatellite-enriched library method and 454 GS-FLX technique. The set of primers amplified dinucleotide to pentanucleotide repeats, and the number of alleles per locus ranged from one to six, one to 11, and one to nine for V. orsiniana, V. javalambrensis, and V. rosea, respectively. Transferability analyses were performed in 20 species corresponding to 10 different subgenera. Conclusions: These results indicate the utility of the newly developed microsatellites across Veronica subsect. Pentasepalae, which will help in the study of gene flow patterns and genetic structure.

The genus Veronica L. (Plantaginaceae) comprises ca. 450 species, which are grouped into 12 subgenera with between two and 180 species each ( Albach et al., 2004 ;Garnock-Jones et al., 2007 ). It includes some perennials of relative economic importance in ornamental horticulture and others that are well-known widespread weeds. Additionally, several species of Veronica are registered on the International Union for Conservation of Nature Red List ( http://www.iucnredlist.org/ ) and other regional catalogs of endangered plants (e.g., Peñas de Giles et al., 2004 ), or are threatened plants with narrow distribution areas (e.g., Petrova and Vladimirov, 2009 ).
Veronica subsect. Pentasepalae Benth. is a monophyletic diploid-polyploid complex and one of the four subsections currently recognized within the also monophyletic Veronica subgen. Pentasepalae M. M. Mart. Ort., Albach & M. A. Fischer ( Albach et al., 2008 ). This subsection comprises ca. 20 perennial taxa and is represented in the temperate regions of Eurasia with one species in North Africa. The complex seems to be of recent origin and divergence, as many diploid representatives are still extant and short branches are found in the phylogenetic analyses based on ITS and plastid DNA sequence data ( Rojas-Andrés et al., 2015 ). Although the diploid species are characterized by subtle morphological differences, each has been recovered as monophyletic in previous studies. Hybridization and polyploidization are widespread in the group, and several authors ( Lehmann, 1937 ;Scheerer, 1949 ;Rojas-Andrés et al., 2015 ) have concluded that gene fl ow and complex relationships among polyploids and their diploid relatives might exist. Interestingly, some of the diploid and polyploid species belonging to Veronica subsect. Pentasepalae are Mediterranean orophytes that face a high risk of extinction with climate warming and/ or grow in Important Plant Areas (IPAs; IPA online database: http://www.plantlifeipa.org/reports.asp ), regions that display exceptionally rich fl oras of biogeographic interest ( Rojas-Andrés et al., 2015 ). Given that current gene fl ow and introgression may have blurred species limits, particularly in hybrid zones, accurate investigations of gene fl ow patterns within and among Veronica subsect. Pentasepalae populations are necessary for conservation and species delimitation purposes.

METHODS AND RESULTS
Microsatellite development -For the microsatellite library, silica gel-dried leaves of 12 diploid individuals of V. jacquinii Baumg. and V. orbiculata A. Kern. were selected from eight different populations (Appendix 1). Ploidy level was checked using fl ow cytometry. A microsatellite library was prepared by Genoscreen (Lille, France) using a 454 GS-FLX (Roche Diagnostics, Meylan, France) high-throughput DNA sequencer ( Malausa et al., 2011 ). Genomic DNA was extracted using the cetyltrimethylammonium bromide method described in Doyle and Doyle (1987 a See Appendix 1 for locality and voucher information for each population . b Deviations from HWE were not statistically signifi cant (ns) and statistically signifi cant at * P < 0.05, ** P < 0.01, and *** P ≤ 0.001.
AAC, AAG, AGG, ACG, ACAT, and ACTC motifs. A total of 32,052 high-quality sequences were obtained. Analyses of these sequences with QDD software ( Meglécz et al., 2010 ) revealed 3010 sequences with microsatellite motifs, for which 195 pairs of primers were obtained. Given that it is too time consuming and not affordable to check all of the primer pairs obtained, 54 of them with low primer pair penalty and different lengths and repeat motifs were selected. These primers were ordered (Eurofi ns, Ebersberg, Germany) to evaluate polymorphic loci on 12 individuals from the complex V. jacquinii-V. orbiculata . PCRs were performed in a total volume of 15 μ L, which contained 1 × PCR Green GoTaq Buffer (Promega Corporation, Madison, Wisconsin, USA), 0.25 mM of each dNTP (Life Technologies, Carlsbad, California, USA), 0.33 mM of each primer, 0.5 units GoTaq DNA Polymerase (Promega Corporation), and 18.2 ng of DNA template. PCRs used the following conditions: an initial step at 94 ° C for 2 min; followed by 35 cycles of 1 min at 94 ° C, 1 min at 50-58 ° C, and 50 s at 72 ° C; and a fi nal extension of 15 min at 72 ° C. All the reactions were conducted on a Mastercycler pro S thermocycler (Eppendorf, Hamburg, Germany). The PCR products were separated by electrophoresis on a 2.5% agarose gel and sent to Macrogen Europe sequencing service (Amsterdam, The Netherlands). In a second step, those primers that were polymorphic in the V. jacquinii-V. orbiculata complex were tested in two individuals from three species, each from a different clade ( V. orsiniana Ten. [core clade], V. javalambrensis Pau [Iberian clade], and V. rosea Desf. [North African clade]), using the same PCR conditions. Twelve polymorphic primer pairs were selected (see Appendix 2 for additional primers). Following the procedure developed by Schuelke (2000) , the sequence-specifi c forward primers were marked at the 5 ′ end with an M13 tail (5 ′ -TGTAAAACGACGGCCAGT-3 ′ ) (Eurofi ns), which was then labeled with 5-FAM, VIC, NED, or PET fl uorescent dyes ( Table 1 )  and 50 ng of DNA template in a total volume of 15 μ L. Conditions of the PCR amplifi cation were as described above, adding 10 cycles of 1 min at 94 ° C, 1 min at 53 ° C, and 50 s at 72 ° C before the fi nal extension. PCR products were analyzed with GeneMarker AFLP/Genotyping Software version 1.8 (SoftGenetics, State College, Pennsylvania, USA).

Population genetics parameters in three further species from
Veronica subsect. Pentasepalae -The fi rst comprehensive phylogenetic analysis of Veronica subsect. Pentasepalae based on DNA sequence data revealed four main clades each corresponding to a broad geographic area ( Rojas-Andrés et al., 2015 ). Thus, for the characterization of the microsatellite markers, diploid populations corresponding to species from different clades were selected (Appendix 1): V. orsiniana (core clade), V. javalambrensis (Iberian clade), and V. rosea (North African clade). The Central Asian clade was not considered because no material was available. The mean number of alleles per locus, observed and expected heterozygosities, possible deviations from Hardy-Weinberg equilibrium (HWE; Table 2 ) , and tests for linkage disequilibrium between markers in each population were estimated using Arlequin version 3.5.1.2 ( Excoffi er and Lischer, 2010 ).
The number of alleles per locus ranged from one to six, one to 11, and one to nine in the V. orsiniana , V. javalambrensis , and V. rosea populations, respectively. Loci 26, 49, and 52 were monomorphic in V. orsiniana , loci 10 and 52 were monomorphic in V. javalambrensis , and in V. rosea , loci 8 and 13 were monomorphic and locus 49 did not amplify. The observed and expected heterozygosities for all populations are shown in Table 2 . Signifi cant deviation from HWE ( P < 0.05) was seen for loci 8, 10, 13, and 54 in V. orsiniana , for locus 50 in V. javalambrensis , and for loci 10 and 50 in V. rosea . Linkage disequilibrium showed signifi cance levels below 0.05 after false discovery rate (FDR) correction in two pairwise comparisons (pair 20-52 in V. rosea and pair 27-54 in V. orsiniana ).

Cross-amplifi cation in other species from
Veronica subsect. Pentasepalae and 10 subgenera of Veronica -Cross-amplifi cation performed for these 12 polymorphic loci showed successful results within the expected allele size in two additional species from Veronica subsect. Pentasepalae : V. austriaca L. and V. dentata F. W. Schmidt. Tests were also performed for 20 additional species from 10 different subgenera within the large genus Veronica ( Table 3 ) . The tests were carried out with the original PCR protocol. The 12 loci tested in agarose gel showed successful amplifi cation of at least several bands. Six of these (8, 10, 13, 19, 26, and 35) showed good amplifi cation results in most samples.

CONCLUSIONS
A set of polymorphic microsatellite markers for Veronica subsect. Pentasepalae is reported. Amplifi cation success for these markers in the cross-transferability tests extends their potential usefulness to other subgenera. These markers will be useful for investigating genetic parameters, which may provide essential information for the conservation of threatened species, as well as data on the role of interspecifi c hybridization in the evolution of the genus.  c Populations used to generate the data included in Appendix 2.


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Editorial: Influenza Virus Vaccines and Immunotherapies

Influenza virus infections are responsible for billions of dollars of economic burden annually worldwide. Regardless of advances in our understanding of the immune evasion mechanism, the disease remains one of the foremost threats for human being (1). Currently available vaccines and therapeutic agents are not very potent against the deadly flu infections because of the frequent mutations in influenza virus subtypes. The mismatch between the circulating strains and vaccine strains critically affects the success rate of the conventional vaccines and requires continuous monitoring of circulating influenza strains. These collective limitations of existing flu vaccines urgently call for the development of novel vaccines with a wide range of cross-protective immunity (2). The discovery and development of new and improved vaccines have been greatly facilitated by the application of new technologies. The articles included in this research topic explain the key methods of improvement in existing vaccines, therapeutics, and mechanism of protection.

The opening articles of the present topic discuss about the novel methods of improving existing influenza vaccines and enhancing immune responses. Various strategies including use of adjuvants, heterologous prime/boost and unique antigen design have shown to induce protective influenza-specific neutralizing antibodies. Lofano et al. reviewed recent advancements in the flu vaccine development and highlight the role of B-cells in controlling influenza virus infections (3). Soema et al. discussed the recent developments and design of T-cell based vaccines based on novel peptide and protein-based vaccine formulations (4). Adjuvants play a critical role in the induction of rapid, effective, and durable immune responses when administered with vaccine antigens. Recent progress in the adjuvant formulations allows existing vaccines to reduce the number of booster doses, increase dose-sparing ability, induce potent T and B-cell responses and enhance breadth of the immune responses against heterotypic antigens (5). Vogel and Brown reported that only single dose of CpG adjuvanted influenza vaccine can induce robust memory T-cell responses and confer protection against heterosubtypic challenge (6). Similarly, in the presence of oil-in-water emulsion adjuvant system (AS03), even a very small dose of influenza vaccine can induce significantly strong immune responses (7). Alternative strategies of immune protection such as use of immuno-modulatory agents like TLR agonist can activate protective non-specific antiviral immune responses. Mifsud et al. demonstrated that mice pre-treated with TLR-2 agonist PEG-Pam2Cys were able to mount specific B- and T-cell responses and also protect mice against heterologous virus challenge (8). These types of alternative strategies can provide immediate immune protection in the absence of effective vaccine without compromising the antiviral specific immunity.

The next generation universal influenza vaccine targets the most conserved structure of the virus and hence confers heterotypic protection, for example, new vaccine strategies target stalk of the HA instead of the globular head. Mallajosyula et al. designed a potent immunogen comprised of HA-stem-fragment from H3N2 strain (A/Hong Kong/1/68) and trimerization motifs: coiled-coil isoleucine zipper and globular β-rich. Immunization of mice with the immunogen induced cross-reactive antibodies and provided only partial protection against homologous virus challenge (9). Li et al. engineered a dual vaccine by incorporating botulinum neurotoxin A (BoNT/A) receptor-binding subdomain in universal influenza vector based on PR8 strain of influenza virus. This vaccine induced protective immunity against both BoNT/A and Influenza virus. Potent immune responses can also be generated by exploiting receptors on antigen presenting cells (APCs) (10). Grodeland et al. demonstrated that a novel DNA vaccine candidate specifically induced Th2 and IgG1 antibody responses or Th1 and IgG2a responses by targeting hemagglutinin to MHC class II molecules or chemokine receptors (XCR1 or CCR1/3/5), respectively (11).

The historical 1918 influenza pandemic caused very high mortality in adult population and the immunological parameters in this population still remain speculative. In light of current understanding of influenza immuno-pathogenesis, McAuley et al. claimed that, the high mortality rate in the adult population is due to the dysfunctional or excessive cross-reactive memory T-cells, induced by previous influenza infections, which render these individual susceptible to the 1918 pandemic influenza (12). Peng et al. provided the evidence that pre-existing seasonal influenza virus HA-specific cross-reactive T-cells can be boosted by a heterologous vaccine (13). Pregnant women are at paramount priority for influenza vaccination due to influenza virus related complications during the pregnancy. Modulation of maternal immunity during the pregnancy can influence the influenza vaccine specific immune responses. Kay and Blish summarize the immunogenicity and efficacy of the influenza vaccine and discuss impact on T and B-cell responses during the pregnancy (14).

Apart from prophylactic vaccine, therapeutic approaches also required for those severely infected with the influenza virus. The use of antibodies seems to be an attractive immunotherapeutic approach for the treatment of various infectious diseases. Sasisekharan et al. reviewed different antibody based strategies to prevent and treat the influenza infections (15). The cells of innate immune system are triggered by signaling pathways during influenza virus infection that causes extensive damage to lung tissues and in airway lining, resulting in severe immunopathology. Ramos and Sesma suggest that these damages can be minimized by selectively modulating the innate signaling pathways using immuno-modulatory drugs while maintaining the ability of the host cells to mount an antiviral response to control virus replication (16). Further, Graham et al. described the role of mast cells in immunopathology during influenza A virus infection and suggested them as a potential drug target in viral infections (17).

The new strategies for vaccine design also require more sensitive and efficient methods for the evaluation of the vaccine potency. Carnell et al. reviewed comprehensive methods for influenza virus neutralization assays, based on the pseudotype viral particles, which utilize chimeric viruses bearing influenza glycoproteins, and depict how such assays can replace the traditional HA neutralization assays for the evaluation of new age influenza vaccines (18). To detect the incidence of Influenza virus with high efficiency and accuracy, Rajput et al. generated HA specific high affinity recombinant single chain variable fragment (scFv) antibodies showing high sensitivity (83.9%) and specificity (100%) for three different strains of influenza virus (19).

The compilation of research articles included in this research topic should help reader to have an overview of different strategies for improving existing influenza vaccines and immunotherapies. This research topic also highlights the progress made in understanding of the immune protection and pathogenic mechanisms of influenza virus.

Influenza virus infections are responsible for billions of dollars of economic burden annually worldwide. Regardless of advances in our understanding of the immune evasion mechanism, the disease remains one of the foremost threats for human being (1). Currently available vaccines and therapeutic agents are not very potent against the deadly flu infections because of the frequent mutations in influenza virus subtypes. The mismatch between the circulating strains and vaccine strains critically affects the success rate of the conventional vaccines and requires continuous monitoring of circulating influenza strains. These collective limitations of existing flu vaccines urgently call for the development of novel vaccines with a wide range of cross-protective immunity (2). The discovery and development of new and improved vaccines have been greatly facilitated by the application of new technologies. The articles included in this research topic explain the key methods of improvement in existing vaccines, therapeutics, and mechanism of protection.
The opening articles of the present topic discuss about the novel methods of improving existing influenza vaccines and enhancing immune responses. Various strategies including use of adjuvants, heterologous prime/boost and unique antigen design have shown to induce protective influenzaspecific neutralizing antibodies. Lofano (6). Similarly, in the presence of oil-in-water emulsion adjuvant system (AS03), even a very small dose of influenza vaccine can induce significantly strong immune responses (7). Alternative strategies of immune protection such as use of immuno-modulatory agents like TLR agonist can activate protective non-specific antiviral immune responses. Mifsud et al. demonstrated that mice pre-treated with TLR-2 agonist PEG-Pam2Cys were able to mount specific B-and T-cell responses and also protect mice against heterologous virus challenge (8). These types of alternative strategies can provide immediate immune protection in the absence of effective vaccine without compromising the antiviral specific immunity.
The next generation universal influenza vaccine targets the most conserved structure of the virus and hence confers heterotypic protection, for example, new vaccine strategies target stalk of the HA instead of the globular head. Mallajosyula et al. designed a potent immunogen comprised of HA-stem-fragment from H3N2 strain (A/Hong Kong/1/68) and trimerization motifs: coiled-coil isoleucine zipper and globular β-rich. Immunization of mice with the immunogen induced crossreactive antibodies and provided only partial protection against homologous virus challenge (9).

Li et al. engineered a dual vaccine by incorporating botulinum neurotoxin A (BoNT/A) receptor-binding subdomain in universal influenza vector based on PR8 strain of influenza virus. This vaccine induced protective immunity against both BoNT/A and Influenza virus. Potent immune responses can also be generated by exploiting receptors on antigen presenting cells (APCs) (10).
Grodeland et al. demonstrated that a novel DNA vaccine candidate specifically induced Th2 and IgG1 antibody responses or Th1 and IgG2a responses by targeting hemagglutinin to MHC class II molecules or chemokine receptors (XCR1 or CCR1/3/5), respectively (11).
The historical 1918 influenza pandemic caused very high mortality in adult population and the immunological parameters in this population still remain speculative. In light of current understanding of influenza immuno-pathogenesis, McAuley et al. claimed that, the high mortality rate in the adult population is due to the dysfunctional or excessive cross-reactive memory T-cells, induced by previous influenza infections, which render these individual susceptible to the 1918 pandemic influenza (12). Peng et al. provided the evidence that pre-existing seasonal influenza virus HA-specific cross-reactive T-cells can be boosted by a heterologous vaccine (13). Pregnant women are at paramount priority for influenza vaccination due to influenza virus related complications during the pregnancy. Modulation of maternal immunity during the pregnancy can influence the influenza vaccine specific immune responses. Kay and Blish summarize the immunogenicity and efficacy of the influenza vaccine and discuss impact on T and B-cell responses during the pregnancy (14).
Apart from prophylactic vaccine, therapeutic approaches also required for those severely infected with the influenza virus. The use of antibodies seems to be an attractive immunotherapeutic approach for the treatment of various infectious diseases. Sasisekharan et al. reviewed different antibody based strategies to prevent and treat the influenza infections (15). The cells of innate immune system are triggered by signaling pathways during influenza virus infection that causes extensive damage to lung tissues and in airway lining, resulting in severe immunopathology. Ramos and Sesma suggest that these damages can be minimized by selectively modulating the innate signaling pathways using immuno-modulatory drugs while maintaining the ability of the host cells to mount an antiviral response to control virus replication (16). Further, Graham et al. described the role of mast cells in immunopathology during influenza A virus infection and suggested them as a potential drug target in viral infections (17).
The new strategies for vaccine design also require more sensitive and efficient methods for the evaluation of the vaccine potency. Carnell et al. reviewed comprehensive methods for influenza virus neutralization assays, based on the pseudotype viral particles, which utilize chimeric viruses bearing influenza glycoproteins, and depict how such assays can replace the traditional HA neutralization assays for the evaluation of new age influenza vaccines (18). To detect the incidence of Influenza virus with high efficiency and accuracy, Rajput et al. generated HA specific high affinity recombinant single chain variable fragment (scFv) antibodies showing high sensitivity (83.9%) and specificity (100%) for three different strains of influenza virus (19).
The compilation of research articles included in this research topic should help reader to have an overview of different strategies for improving existing influenza vaccines and immunotherapies. This research topic also highlights the progress made in understanding of the immune protection and pathogenic mechanisms of influenza virus.