Here,we show for the first time that the chemokine RANTES/CCL5 binds to the surface ofhuman endothelial cells in a regular filamentous pattern. Furthermore, the filamentsbound to the surface in a heparan sulfate-dependent manner. By electron microscopywe observed labeling for RANTES on membrane projections as well as on the remainingplasma membrane. Mutant constructs of RANTES restricted either in binding toheparin, or in forming dimers or tetramers, appeared either in a granular,non-filamentous pattern or were not detectable on the cell surface. The RANTESfilaments were also present after exposure to flow, suggesting that they can bepresent in vivo. Taken together with the lacking in vivo or invitro effects of RANTES mutants, we suggest that the filamentous structuresof RANTES may be of physiological importance in leukocyte recruitment.Vascular endothelial cells present luminal chemokines that arrest rolling leukocytesby activating integrins. It appears that several chemokines must form higher-orderoligomers to elicit proper  At sites of inflammation, activated endothelial cells present luminal adhesion moleculesand chemokines to recruit circulating leukocytes. A crucial step in this process is thearrest of rolling leukocytes that is triggered by chemokines and mediated by integrinactivation23In vitro, several chemokines bind to GAGssuch as heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate544RKNR47, located on the surface exposed 40s loop ofthe protein1144AANA47) results in a variant with asubstantially reduced heparin affinity1113Given that soluble chemokines would be rapidly washed away by the blood flow, chemokinesare thought to be immobilized at the luminal surface of endothelial cells through lowaffinity interactions with sulfated glycosaminoglycan chains (GAGs) ofproteoglycans56Another particular feature of RANTES is its propensity to oligomerize into higher-ordercomplexes at high concentrations, high pH or in the presence of GAG15137In the present study, we have analysed the distribution of endogenous and recombinantRANTES on vascular endothelial cells. We show that secreted RANTES bound at the cellsurface, not in a diffuse or aggregated pattern, but surprisingly as specific, wellordered filaments that elongated over time. The filamentous expression of RANTES was notfound when expressing mutants that are restricted to form dimers or tetramers, or whenexpressing a mutant with reduced GAG affinity. These data add further support to themerging view that formation of higher order oligomers of RANTES is crucial to elicit itsfully biological function in activation of leukocytes.et al.,unpublished data). When HUVECs were cultured in vitro and stimulated withTNFα in combination with IFNγ before fixation and immunostaining, RANTESmainly localized in elongated, filamentous structures  is suggested to be important foroligomerization as R44 may exert stabilization forces to the dimer interface,and R47 is shown to interact with the neighbouring molecule in RANTESstructures16RANTES can self-associate and form higher-order oligomers in aconcentration-dependent manner5163244AANA47, or E66A. We also examinedthe mutant Y3A which shows in vivo properties similar to44AANA47 but has unknown oligomerizationstatus44AANA47, and E66A revealed apattern substantially different from that of wild type (wt) RANTES  RANTES . The mutstaining . Only 10staining . The mutstaining .44AANA47 were found in approximatelytwo-thirds and half the amount, respectively, of that measured for wt  showed a filamentous RANTES pattern (data not shown). Moreover, weelectroporated HUVECs with low amounts of plasmid DNA encoding wtRANTES(4 μg compared to 20 μg in the standard protocol) whichresulted in a dramatic reduction in protein expression and fewer positive cells.Despite the low level, wtRANTES organized in filaments (data not shown).Finally, we performed ELISA on supernatants harvested from HUVECs electroporatedwith plasmid DNA encoding the various constructs. Both Y3A and E66A were presentin higher amounts than wt, whereas E26A andd for wt . Altoget44AANA47 and E66A.Next, to examine whether the mutant constructs of RANTES were present on theendothelial cell surface or restricted to intracellular compartments, weimmunolabelled live HUVECs kept on ice and compared the pattern towards that offixed and permeabilized cells. As shown in Pre-existing tetraspanin-enriched microdomains (TEMs) containing adhesionreceptors such as ICAM-1, VCAM-1, and CD44 are suggested to function as adhesiveplatforms on the endothelial cell surface3535382 as the force has been shown to supportadhesion of both monocytes and lymphocytes40in vivo  failed to appear asfilaments on cultured endothelial cells.Several lines of evidence support a role for higher-order, oligomerized chemokines inleukocyte recruitment. First, wtRANTES but not disaggregated mutants, recruitsleukocytes to the peritoneal cavity13143444AANA47 did not formfilaments  fits wellwith its rapid appearance in peripheral blood after intraperitoneal injection44AANA47-RANTES added to cultures of humanmicrovascular endothelial cells does not bind to the cell surface44RKNR47 constitutes the principal GAG-bindingsite58Our observation that the mutant in vitro under flowconditions, the E26A mutant showed reduced potency compared to the wtRANTES. Themutation E26A reduced the number of monocytes that arrested to the endothelialmonolayer, and the authors suggested that RANTES oligomers are required to bridgesurface-bound RANTES and CCR1In our experiments, filament formation generated by the tetramer-restricted mutantE26A was reduced by more than 90% compared to the wt. Following injection into themouse peritonitis model, E26A and wtRANTES appeared to be equally efficient inrecruitment of cells, and it was concluded that the smallest leukocyte-recruitingform of RANTES is a tetramer44AANA47 the inability to recruit leukocytes ininflammation and the ability to inhibit cell recruitment induced by wtRANTES44AANA47 variant,Y3A binds heparin. We found that Y3A, similar to E26A, appeared in a granularpattern on the endothelial cell surface, indicating that its ability to formoligomers is abolished. Finally, the E66A mutant which forms dimers, was detectedintracellularly and was secreted in similar amounts to that of Y3A. Recombinant E66Awas reported to bind to the surface of human microvascular endothelial cellsThe mutant Y3A shares with the heparin-deficient mutantet al. proposed a model for how RANTES organizes intooligomers based on detailed structural analyseset al. observed long oligomers at pH 7, and suggested that binding tosulfated GAGs would further promote length, as negatively charged GAG can neutralizeelectric repulsion forces between RANTES dimersInterestingly, Wang et al. found thatleukocytes interacted with both the long and short projectionsDuring the initial events of leukocyte extravasation, membrane projections wouldprobably increase the accessibility of the endothelium towards that of theleukocyte. Luminal endothelial membrane projections have been given names such asmicrofolds, microvilli, filopodia, protrusions, and nanotubes82223et al., unpublished data). This pattern wasreminiscent of the ICAM-1 distribution observed after immunofluorescence, and ourimmunofluorescent data are very similar to what has been reported by others withICAM-1 being present on microvilli or microvilli-like projections383947Upon binding to leukocyte chemokine receptors, chemokines can trigger complexsignaling transduction cascades leading to activation of integrins and, ultimately,to adhesion via binding to adhesion molecules such as ICAM-1et al., unpublished data). In our studies thefilaments were unique to RANTES, and due to high positive charge, it is proposedthat RANTES is hindered from forming the more common organization of chemokine highorder oligomers, i.e. globular complexes716in vivo,our data imply that presence of filaments may vary between types of endothelialcells, the tissue site, and inflammatory status.We have stained for numerous chemokines after cytokine-stimulation or transfection ofchemokine-encoding DNA into cultures of endothelial cells20in vivo. Moreover, that filaments of RANTES can be of functionalimportance is supported by a study that shows reduced ability of the RANTES tetramerE26A to arrest monocytes on endothelial monolayersetal. suggested that oligomerization of chemokines might be important forthose whose GAG binding sites overlap with the receptor binding sites, as is thecase for RANTES, MCP-1 and MIP-1β so that while some chemokine subunits bind toGAGs others can be exposed to the receptor742505153In conclusion, our data together with biochemical analysesFetal bovine serum (FBS), gentamicin, fungizone, L-glutamine, MCDB 131, andOpti-MEM I were purchased from Life Technologies , and trypsin-EDTAwas from Bio-Whittaker . Recombinant human TNFα andIFNγ, recombinant human epidermal growth factor (EGF), recombinant humanbasic fibroblast growth factor (bFGF), recombinant human MCP-1, mouse and goatanti-human RANTES antibodies were purchased from R&D Systems . A second mouse anti-humanRANTES antibody was a kind gift from M. Sticherling , and mouse anti-humanRANTES clone VL1 was from Biosource . Rabbit anti-human RANTES(500-P36) was from Peprotech , and mouse anti-heparan sulfate,clone 10E4, and hyaluronan binding protein was from Seikagaku Corporation. The secondary rabbit anti-goat antibody used for electronmicroscopy was from DAKO . The fluorescent secondary anti-mouse or anti-rabbit antibodies were from Molecular Probes, fluorescein ulex europaeus agglutinin I, andbiotinylated horse anti-mouse IgG from Vector Laboratories ,streptavidin-Cy2 from Amersham Pharmacia Biotech , andstreptavidin-Cy3 from Jackson . Protein A coupled to goldparticles of different sizes was purchased from George Posthuma . Heparin was from LeoPharma . All other reagentswere from Sigma Chemical .AAGCTTACCATGAAGGTCT-3' and reverse5'- AGAATCTAGACTAGCTCATCTCCAAAGAGTTGATGTACT-3') were designedto introduce HindIII and XbaI restriction sites (underlined),respectively, that were used to clone the RANTES-encoding DNA fragment into thepcDNA3.1(+) vector . Based on this construct, alanineexchange of selected amino acids was performed using the QuikChangeTMSite-Directed Mutagenesis Kit  according to theinstructions by the manufacturer. The following primers were used to introducethe mutations, giving the sequence of the sense primers with mutated nucleotideswritten with small letters: Y3A:5'-CTGCATCTGCCTCCCCAgcTTCCTCGGACACCACACC-3'; E26A:5'-CCCGTGCCCACATCAAGGcGTATTTCTACACCAGTGGCAAGTG -3'; 44AANA47:5'GCAGTCGTCTTTGTCACCgcggcGAACgcCCAAGTGTGTGCCAACC -3'; E66A: 5'-CGGGAGTACATCAACTCTTTGgcGATGAGCGCGGTACCG -3'. The corresponding antisenseprimers were complement reverse.RANTES was amplified from cDNA derived from TNFα/IFNγ-activated HUVECs.Primers . Human umbilical vein endothelial cells (HUVECs) were isolated asdescribed by JaffeHUVECs were trypsinized, washed and resuspended in OptiMEM I containing 2.5% FBS,before transfection by electroporation using 20 μg DNA according tothe protocol 0394 from BTX . Following electroporation, the cellswere cultivated for approximately 24 h before fixation or staining ofcell surface associated RANTES.To examine whether RANTES was bound to the surface in a GAG dependent manner,monolayers of HUVECs were stimulated with 10 ng/ml TNFα combined with1 ng/ml IFNγ for 48 h before incubation with a mixture ofheparinase I, II, and III (0.5 U/ml) for 2 h at 30°C andsubsequently fixed and labelled for RANTES as described above.Streptomyces hyaluronidase  was added directly to cell cultures to yield afinal concentration of 20, 50, or 100 μg/ml and 20 TRU/ml,respectively, and incubation continued for 3 hr at 37°C in aCO2-containing atmosphere.Sterile testicular hyaluronidase or HUVECs were electroporated with wtRANTES and cultivated on gelatine-coatedchamberslides (Lab-Tek) for approximately 24 h before addition of100 ng/ml TNFα . After 12 h ofstimulation, the cells were cultivated with 200 ng/ml recombinant humanMCP-1 for 20 min. The cultures were then washed and peripheral bloodmononuclear cells were added to the HUVECs 20 min before fixation.Mononuclear cells were isolated by centrifugation on Lymphoprep according toinstructions of the manufacturer .HUVECs were cultivated on gelatine (1% (w/v)) coated 10 × 10 mmglass coverslips or Lab-Tek chamber slides . Forlabelling of permeabilized HUVECs, cells were fixed in 4% paraformaldehyde for10–15 min before washing in phosphate-buffered saline (PBS). Forimmunostaining, the fixed monolayers were permeabilized with 0.05% saponinbefore incubation with the antibodies, alternatively, 0.1% saponin was includedin all solutions. In another set of experiments, recombinant RANTES was added togelatine (1% (w/v)) coated chamber slides in the absence of HUVECs for35 h. RANTES was incubated in endothelial cell growth medium MCDB 131without FBS or in conditioned MCDB 131 with FBS. Alternatively, heparin was added in different concentrations beforefixation. After sequentially labelling with primary antibodies and secondaryreagents, the slides were mounted in mowiol combined with DABCO or polyvinylalcohol. Labelling of cell surface-associated RANTES was performed on ice withcold (4 °C) antibody solution. The primary biotinylated antibody was addedfor 45 min before fixation in paraformaldehyde followed by washing in PBSand sequential labelling with streptavidin-Cy3 or anti-rabbit Cy2. Theimmunostained cells were examined by an Axioplan 2 imaging Zeiss microscopeusing Plan-NEOFLUAR 40× and 100× oil-objectives or a confocal laserscanning microscope  with A Plan apochromat100×/1.4 oil objective equipped with an Ar (488 nm) and a He/Ne(543 and 633 nm) laser. Cells that expressed two or more RANTESfilaments, were defined as filament-forming cells in the experiments shown inHUVECs were grown in 10 cm diameter culture dishes and cytokine-stimulatedfor 36 h before fixation in 0.1 M PBS containing 4%paraformaldehyde alone or a combination of 0.1% glutaraldehyde and 4%paraformaldehyde for 3 h at room temperature. After washing in 1 ×PBS, cells were scraped-off and spun down. Cell pellets were embedded in 1× PBS/12% gelatine and after infiltration with 2.3 M sucroseover-night at 4°C, cut into small blocks, mounted on pins and frozen inliquid nitrogen. Ultrathin cryosections of about 60–70 nm thicknesswere obtained by cutting at −120°C with a Reichert Ultracut Sultracryomicrotome from Leica . Cryosections were picked upin a 1:1 mixture of 2% methylcellulose and 2.3 M sucrose. Cryosectionswere then sequentially incubated with the goat anti-human RANTES antibody, therabbit anti-goat antibody, and protein A-gold particles diluted in 1 ×PBS/1% BSA for 30 min at room temperature with extensive washing betweenthe incubations.  Finally, cryosections were contrasted with a 1:9 mixture of 3%uranyl-acetate and 2% methylcellulose before examination. When quantifying thedistribution of RANTES, 30 pictures were utilized and gold particles associatedwith membrane projections versus the remaining plasma membrane were counted.2 adjusted to 6%.By use of a pump, medium with temperature 37°C was applied to the flowchamber corresponding to shear stress 1 dyne/cm2. The flowrate was calculated by use of the formula T = 3μQ/2ba2, whereT = wall shear stress, μ = coefficient of viscosity (0.7 centipoise), Q =volumetric flow rate (cm3/s), a = half channel height (127× 10−4 cm), and b = channel width(0.8 cm)Glass coverslips were coated with gelatine (1% (w/v)), HUVECs were added andcultivated at standard conditions for 24 h before stimulation forapproximately 40 h with TNFα (10 ng/ml) and IFNγ(1 ng/ml). Next, the cover slips were mounted in a flow chamber which wasplaced on a microscope stage for live imaging. The stage was enclosed by anincubator with temperature 37 °C and COStatistical analyses were performed by use of GraphPad Prism version 6.04.Responses for each experimental group are presented as means with SEM.Differences between groups were analyzed by using one-way ANOVA with theŠidák method for multiple comparisons. p-values < 0.05 wereconsidered significant.I.Ø., N.B., T.B., A.M.K. performed experiments; I.Ø. and N.B. preparedfigures, all authors analyzed data; G.H. initiated the study; I.Ø., N.B.,O.B. and G.H. designed the study, I.Ø. and G.H. wrote the manuscript.Supplementary figures


MICB) encodes a ligand for activating NKG2D that expressed in natural killer cells, γδ T cells, and αβ CD8+ T cells, which is associated with autoimmune diseases, cancer, and infectious diseases. Here, we have established a system for genotyping MICB alleles using allele-specific primer extension (ASPE) on microarrays. Thirty-six high quality, allele-specific extension primers were evaluated using strict and reliable cut-off values using mean fluorescence intensity (MFI), whereby an MFI >30,000 represented a positive signal and an MFI <10,000 represented a negative signal. Eight allele-specific extension primers were found to be false positives, five of which were improved by adjusting their length, and three of which were optimized by refractory modification. The MICB alleles  present in the quality control panel could be exactly defined by 22 allele-specific extension primers. MICB genotypes that were identified by ASPE on microarrays were in full concordance with those identified by PCR-sequence-based typing. In conclusion, we have developed a method for genotyping MICB alleles using ASPE on microarrays; which can be applicable for large-scale single nucleotide polymorphism typing studies of population and disease associations.Major histocompatibility complex (MHC) class I chain-related gene B ( MICB) is expressed as three extracellular domains, a transmembrane segment, and a carboxy-terminal cytoplasmic tail [+ T cells [http://www.ebi.ac.uk/imgt/hla), over 40 polymorphic alleles have been found in exon 2 to 6 of MICB. Specific polymorphisms are associated with autoimmune diseases, cancer, and infectious diseases, as well as with the success of hematopoietic stem cell transplantation. For example, MICB*004 allele is significantly associated with susceptibility to rheumatoid arthritis in Spanish Caucasians [MICB*005:02 allele is negatively associated with cervical cancer in the Thai population [MICB*008 allele  is positively associated with ulcerative colitis in the Han Chinese population of central China [MICB*008 allele is also associated with a reduction in severity of dengue fever [MICB*008 and *002 alleles are associated with susceptibility to celiac disease in Europeans [MICA and MICB alleles matched between the donor and recipient [The product of major histocompatibility complex (MHC) class I chain-related gene B  and PCR-sequence-based typing (PCR-SBT) [MICB alleles by optimizing allele-specific extension primers for ASPE on glass slides.In previous studies, genotyping PCR-SBT) , 10. MetPCR-SBT) –13. In t2, 0.5% Tween 20, and 100 μg/mL Proteinase K) for 60 min at 56°C and then inactivated for 10 min at 95°C. After extraction, the DNA concentration was adjusted to 100 ng/μL and the resulting DNA samples were used as the PCR template in the genotyping assays.Genomic DNA was extracted from peripheral blood samples taken from 200 healthy Korean individuals  using an AccuPrep Genomic DNA Extraction Kit , following the manufacturer’s instructions. Briefly, the cells were lysed in 100 μL K buffer , 2.5 mmol/L MgClMICB genotyping by PCR-SBT. Six samples among them were defined to be MICB*005:01, *005:06 and *024 alleles, which did not find in Korean population and included to establish MICB genotyping on microarrays  and 68 DNA samples for MICA Exchange (MICA#025 ~ 092) were used for roarrays .The study was approved by the Catholic University Institutional Review Board (IRB), with written informed consent from all subjects .MICB sequence without SNPs. Mismatched control extension primers were designed to contain artificial mismatches at the 1st, 2nd, 3rd, and 4th base from the 3′ ends of the N0 extension primers, and were called the N1, N2, N3, and N4 groups, respectively. Each group consisted of four primers containing an adenine, cytosine, guanine, or thymine  as the mismatch at the 3′ end , which were designed based on the sequences of the MICB polymorphisms with an additional 18 bp oligo(dT) spacer at the 5′ end, was dissolved in a buffer containing 350 mmol/L sodium bicarbonate (pH 9.0)  Tables . The pri4 solution containing 1 g NaBH4, 300 mL PBS, and 100 mL EtOH. Finally, the slides were rinsed with 0.2% sodium dodecyl sulfate followed by distilled water and then dried. The processed slides were stored at −70°C in a silica-gel box.Next, to deoxidize the aldehyde residue, the slides were washed for 30 min, first with 0.2% sodium dodecyl sulfate , followed by distilled water, and then with an NaBHMICB polymorphisms, which are present in exon 2, 3, 4, and 5 of the MICB gene. The forward primers were modified to have a phosphate group (P) at the 5′-end. The following primers were used in each single tube: sense 1, 5′-P-CAATGTGAAGTTATTTCCAGGAAGAAG-3′ from positions 7369 to 7395); antisense 1, 5′-CCAGGGTCGGTACCTGTTCT-3′ (from positions 8230 to 8249) for MICB exon 2 and 3; sense 2, 5′-P-CTGTTCCCTGCATCTCCCTTAGA-3′ (from positions 8765 to 8787); antisense 2, 5′-CCCATCTCCAGAAACTGTCCCC-3′ (from positions 9346 to 9368) for MICB exon 4 and 5 [2), 200 μmol/L dATP/dGTP/dCTP, 200 μmol/L dTTP/dUTP (dT:dU = 7:3), 4U Taq DNA polymerase  and 10 μmol/L of each primer. The reactions were amplified using a Mycycler Thermo Cycler  with the following conditions: initial denaturation of 98°C for 20 s; 8 cycles of denaturation at 98°C for 5 s, annealing at 65°C for 30 s, and extension at 72°C 30 s; followed by 37 cycles of denaturation at 98°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; and followed by a final extension at 72°C for 10 min. Then, the reactions were held at 20°C.Primer sets were designed to amplify  4 and 5 . PCR wasThe PCR products were treated with 10× λ exonuclease buffer containing 3.5U λ exonuclease  and 1.75U shrimp alkaline phosphate . After the PCR products had been cleaved into approximately 50-bp fragments, they were hybridized with a mixture that contained 25× Themo Sequenase buffer , 3.5U Thermo Sequenase , and 0.1 mmol/L each of both dATP/dTTP/dGTP and Cy5-dCTP at 60°C for 1 h. To remove non-specific primers, the slides were washed for five minutes with a solution containing 0.1% Alconox and then rinsed twice with distilled water, for 5 min each time.The data were obtained using the EasyScan software after scanning with NanoDscan  and then analyzed for genotyping using Microsoft Excel. The background mean fluorescence intensity (MFI) was subtracted from the MFI of each primer; cut-off values were determined using quality control samples .MICB primers were designed as previously described [2), 200 μmol/L dATP/dGTP/dCTP/dTTP, 0.6 U Taq DNA polymerase , bovine serum albumin, Cresol red (in 28% sucrose) and 3.7 μmol/L of each primer. The cycling conditions were as follows: 95°C for 5 min; then 19 cycles of 94°C for 45 s, 65°C for 45 s (a reduction of −0.5°C per cycle), and 72°C for 2 min; then 13 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 2 min; and finally, 72°C for 10 min. Then, the reactions were held at 20°C.escribed . PCR wasMICB exon 6 primers were designed to differentiate MICB*005:02 allele from MICB*010 allele in exon 6, with the forward primer as 5′-GGGCAACTGAAGAGAGAAAAG-3′ and the reverse primer as 5′-CAGGAGCAGTCGTGAGTTTG-3′. The PCR conditions were as follows: an initial denaturation of 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; and a final extension at 72°C for 10 min. Then the reactions were held at 20°C.The MICB primers for PCR amplification were also used as the sequencing primers. The sequencing data were analyzed using the ABI Factura software and the ABI Sequence Navigator program .Purified PCR products were sequenced using an ABI 377 DNA sequencer; the The melting temperature (Tm) for sequences longer than 13 bases was calculated as follows:W, x, y, and z indicate the number of adenine, thymine, guanine and cytosine , respectively, in the sequence.t-test. We corrected for statistical analyses calculated from five or less observed scores using Fisher’s exact test.The Tm and GC content in primers from the successful group of allele-specific extension primers were compared to those from the failed group using the unpaired Student’s MICB exon 2, 3, 4, and 5. The primers were 19 bp long, with a Tm of 53.0 ± 3.0°C, and a GC content of 57.4 ± 7.4% . The PCR products of MICB exons 2–5 did not showed any positive signals on the MICA allele-specific extension primers of microarray for MICA allele genotyping [The ability to discriminate notyping .MICB alleles previously identified in Koreans and other populations were selected as the quality control panel for ASPE on microarrays. We defined MICB alleles of 163 samples in the UCLA International KIR and MICA Exchange Report program. Among them, six samples including three MICB alleles of MICB*005:01, *005:06 and *024 alleles which did not find in Korean population were added in MICB alleles of the other 157 samples were also found in Korean population. A validation of the amplification reaction has been performed for the alleles  included in the quality control panel. To identify the 41 known MICB alleles, 36 allele-specific extension primers were designed with a length of 18 to 21 bases. Among these primers, the specificity and sensitivity of only 22 primers could be determined by the quality control panel used in this study (P < 0.02 for each category).Eighteen samples including 13 is study . Of thesMICB*002:01, *003, *005:02/*010, *005:03, *008, *009N, *018, and *024 alleles present in our panel showed perfect matches by 22 allele-specific extension primers validated with the quality control panel after optimal modification of primers , followed by MICB*005:02/*010/*004:01 (n = 32), *005:02/*010/*002:01 (n = 30), *005:02/*010/*005:03 (n = 16), *005:02/*010/*008 (n = 15), *005:02/*010/*009N (n = 8), *005:02/*010/*003 (n = 8), *002:01/*014 (n = 6), *002:01/*008 (n = 4), *004:01/*004:01 (n = 4), *002:01/*004:01 (n = 4), *002:01/*005:03 (n = 2), *003/*008 (n = 2), *005:03/*009N (n = 2), *002:01/*003 (n = 2), *005:02/*010/*005:01 (n = 2), *005:02/*010/*005:06 (n = 2) and*002:01/*002:01 (n = 2). The other 9 combinations were seen in only one sample each . Because this MICB alleles’ genotyping on microarrays with ASPE can discriminate the 18 SNPs among 28 SNPs known in MICB gene . However, we showed MICB*005:02/*010 allele because the primer sets used to amplify templates for ASPE on microarrays were not designed to differentiate MICB*005:02 allele from MICB*010 allele in exon 6. MICB*018 and *019 alleles, which have not previously been reported in Koreans, were found in this study.Genotyping by ASPE on microarrays was fully concordant with mparison . MICB*002+ concentration [The ASPE method depends for identifying alleles on the sequence-specific extension of immobilized allele-specific extension primers that differ at the 3′ ends of their SNPs . Howeverntration . Of the Taq polymerase offers the greatest template discrimination (40- to 100-fold) against mismatches to thymine, guanine, or cytosine  at the 3′ end of a primer, but not against mismatch to adenine (A) [Allele-specific PCR with nine (A) . To imprnine (A) . For priDifferent approaches have been proposed to improve the specificity of ASPE. As the specificity of primer-directed extension is not sufficient for quantitative SNP analysis, artificial mismatched bases have been introduced into the 3′ end regions of the specific primers as a way of improving the switching characteristics of the primer extension reactions. The best position in the primer for such artificial mismatched bases is the third position from the 3′ end of the primer . In our MICB alleles, only 22 primers could be defined or improved using the control panel in this study. Therefore, we could not define the other rare alleles and it is necessary to confirm the quality of the 14 remaining primers using samples from other populations. In Korean population, the MICB alleles detected by the genotyping microarray were fully in concordance with those detected by PCR-SBT and we compared the MICB population allele frequency (Korean population) with the test sample frequency in MICB*018 and *019 alleles, had not been previously found in Koreans [MICB alleles in this study using verified allele-specific extension primers. Our microarray did not include SNP in exon 6 because these SNPs are present only in very rare alleles which have been not reported in population data of published papers and are not present even in 163 samples of the UCLA International Exchange Report program. Although we designed primers to detect SNPs in exon 2 to 5 of MICB, new alleles have recently been defined by SNPs in exon 6 [MICB*010 and *005:02 alleles could not be discriminated by ASPE on microarrays because the primers had not been designed to differentiate MICB*005:02 allele from MICB*010 allele in exon 6.Although we designed 36 primers to detect almost all of the known 41  Koreans . Therefon exon 6 . MICB*01MICB alleles identified by this method were MICB*002:01, *003, *004:01, *005:01, *005:02/*010, *005:03, *005:06, *008, *009N, *014, *018, *019 and *024 Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.


Haematococcus pluvialis has emerged as a promising biomass feedstock for the ketocarotenoid astaxanthin and neutral lipid triacylglycerol. Motile flagellates, resting palmella cells, and cysts are the major life cycle stages of H. pluvialis. Fast-growing motile cells are usually used to induce astaxanthin and triacylglycerol biosynthesis under stress conditions (high light or nutrient starvation); however, productivity of biomass and bioproducts are compromised due to the susceptibility of motile cells to stress. This study revealed that the Photosystem II (PSII) reaction center D1 protein, the manganese-stabilizing protein PsbO, and several major membrane glycerolipids , decreased dramatically in motile cells under high light (HL). In contrast, palmella cells, which are transformed from motile cells after an extended period of time under favorable growth conditions, have developed multiple protective mechanisms—including reduction in chloroplast membrane lipids content, downplay of linear photosynthetic electron transport, and activating nonphotochemical quenching mechanisms—while accumulating triacylglycerol. Consequently, the membrane lipids and PSII proteins (D1 and PsbO) remained relatively stable in palmella cells subjected to HL. Introducing palmella instead of motile cells to stress conditions may greatly increase astaxanthin and lipid production in H. pluvialis culture.The unicellular microalga  Haematococcus pluvialis can accumulate the largest amounts [up to 4% of its dry weight (DW)] under various adverse environmental or culture conditions H. pluvialis in photobioreactors has been exploited to produce natural astaxanthin H. pluvialis is an alternative solution for removing CO2 from fossil-fired power plants Astaxanthin is a superb antioxidant and a natural food coloring agent that has been used in nutraceutical, aquaculture, and poultry industries H. pluvialis2+ to the cultures H. pluvialis cells to adverse culture conditions leads to a substantial reduction in biomass productivity, a major obstacle that prevents expansion of the H. pluvialis industry.A two-stage cultivation strategy is often applied to mass culture of H. pluvialis strain CCAP 34/12, which is dominated by flagellates at the exponential growth phase, was more susceptible to HL stress than another strain (SAG 34/1b) dominated by resting vegetative cells. These resting cells are also called palmella cells and are transformed from flagellates under favorable growing conditions. The death of flagellates under HL was attributed to the production of reactive oxygen species (ROS) Haematococcus strains. A recent comparative proteomic analysis of flagellates and resting  cells from a single Haematococcus strain showed that a number of proteins involved in stress responses were induced in the resting cells but absent in the flagellates It has recently been observed that the Haematococcus cells cope with HL. To gain more insight into the molecular–level changes in lipids that occur in response to HL, we developed a mass spectrometry–based lipidomics method for absolute quantification of glycerolipids. Our results suggest that introducing resting palmella instead of motile flagellates into mass culture represents a promising strategy to increase the production of biomass and bioproducts from H. pluvialis.The aim of this study was to determine the physiological and biochemical changes that occur during the transformation of motile flagellates into resting palmella cells and to dissect the key mechanisms by which the different forms of Haematococcus pluvialis NIES144 was obtained from the National Institute for Environmental Studies in Tsukuba, Japan. Algal cells were grown in 2.8-L flasks containing 1 L basal growth medium −2 s−1). Cultures were maintained in a resting culture mode with manual shaking once per day. To obtain motile cells, cultures were maintained under the above culture conditions for 4 days, at which point ca. 70% of cells were motile cells and the remainder were palmella cells; the flagellates suspended in the growth medium were then harvested and enriched with 98.0±1.8% motile cells. Palmella cells were predominant in the cultures maintained under the above conditions for 7 days. To increase the quantity of palmella cells, cells that settled at the bottom of the flasks were collected and washed with fresh growth medium five times to remove any remaining motile cells. Motile and palmella cells were both exposed to continuous high light (HL) illumination (150 µmol photons m−2 s−1) for 24 h. Red cyst cells transformed from motile cells are referred to as RC-M, and those induced from palmella cells are termed RC-P. Algal cells were harvested by centrifugation at 1,000 g for 10 min. Aliquots of fresh cell pellets were resuspended in the breaking buffer  for immunoblotting, or growth medium for chlorophyll fluorometry analysis. Freeze-dried algal biomass were used for biochemical composition  analyses. Each type of cells was prepared in triplicate.β-carotene, chlorophyll a (chla), and chlorophyll b (chlb) contents were analyzed by high-performance liquid chromatography (HPLC) according to the method described previously Canthaxanthin, astaxanthin, a fluorimetric analysis was conducted using the Dual-PAM-100 system . Harvested cells were resuspended in fresh growth medium and were dark-adapted for 15 min before measurement. One mL samples were loaded into 1 cm cuvettes and stirred gently with a magnetic stir bar. The minimal fluorescence (F0) was recorded under the measuring light, and after ∼5–10 s, a saturated pulse light  was applied to fully close the PSII reaction centers to measure the maximum fluorescence (Fm) −2 s−1) were switched on, and at intervals of 35 s, saturating flashes were applied. From this Saturation Pulse analysis, the maximum fluorescence in the light (Fm′) and steady-state value of fluorescence (F) were recorded. The following parameters indicating photosynthetic efficiency and energy quenching at PSII were calculated: Fv/Fm   =  (Fm−F0)/Fm; Y(II) (quantum yield of PSII)  =  (Fm′−F)/Fm′; ETR(II)  =  Photosynthetically active radiation×0.5×0.84×Y(II); NPQ   =  (Fm−Fm′)/Fm′; and Y(NO) (non-regulated energy dissipation)  =  F/Fm Pulse amplitude modulated chl2CO3, 40 µL 30% sucrose, and 5% SDS and were then vortexed at 3,000 rpm for 30 min at room temperature to extract total proteins. Insoluble proteins were removed by centrifugation at 12,000 g for 10 min at 4°C. The concentration of total membrane proteins in the supernatant was measured with a CB-X protein assay kit . Proteins were separated by SDS-PAGE  and transferred to nitrocellulose membranes. Primary antibodies of the D1 protein of PSII, Rieske iron-sulfur protein (RISP) of cytochrome b6f complex, PsaA protein of PSI, and PsbO protein of the oxygen-evolving complex (OEC) were obtained from Agrisera (Sweden). Antigen-antibody complexes were visualized using an enhanced chemiluminescence substrate detection kit . Intensities of visualized protein bands were measured by using the program ImageJ (http://imagej.nih.gov/ij/).Algal cells were disrupted using a mini bead-beater . The homogenates were centrifuged at 1,000 g for 3 min at 4°C to remove unbroken cells and cell debris. The supernatant was then transferred to a new tube and centrifuged at 12,000 g for 30 min at 4°C to obtain the crude membranes. The resulting pellets were resuspended in 60 µL SBA buffer containing 0.1 M dithiothreitol, 0.1 M NaTen mg algal biomass was incubated with 100 µL 1 M NaOH at 80°C for 10 min and then diluted with 900 µL water. Insoluble cell debris was removed by centrifugation at 12,000 g for 30 min at 4°C and was then extracted twice. The resulting supernatants from three extractions were combined for protein assays using a Bio-Rad Protein Assay . Bovine serum albumin was used for calibration.Ten mg algal biomass was pretreated with 0.5 mL acetic acid for 20 min at 60°C to break the cell walls. Samples were immediately placed on ice, and 10 mL acetone was added to each sample to extract the pigments. The extracts were centrifuged at 1,000 g for 2 min at 4°C. The resulting pellets were resuspended in 4 mL trifluoroacetic acid and incubated in boiling water for 4 h to further disrupt the cells and extract the carbohydrates. Samples were cooled on ice before 5 mL water was added. Cell debris was removed by centrifugation at 10,000 g for 2 min at 4°C, and the supernatant was recovered for total carbohydrate measurement using the phenol-sulfuric acid method v/v) -1 at 200°C; spray capillary voltage, 4,000 V for the positive ion mode and 3,500 V for the negative ion mode; gas temperature, 250°C; gas flow, 5 mL min−1; and sheath gas temperature, 350°C. Chloroplast membrane lipids, including monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), were identified by precursor ion scanning for lipid ions, which yielded the diagnostic ions associated with their head groups induced by collision 10H22NO5]+ (m/z 236) was used for precursor ion scanning. TAGs were identified using sequential neutral loss scanning For lipidomic analysis, 10 mg lyophilized algal cells were homogenized in liquid nitrogen, and lipids were extracted with chloroform∶methanol  of DGTS, PC, and PE were detected by multiple reaction monitoring (MRM) in a positive ion mode, while the ammonium adducts ([M+NH4]+) of MGDG, DGDG, and TAG were analyzed by single-stage mass spectrometry (MS). The deprotonated forms of the anionic glycerolipids PG, PI, and SQDG were analyzed by MRM in a negative mode. Prior to MS analysis, lipid extracts were separated on a ZOBAX SB C18 column  for the positive mode or on an Extend C18 column  for the negative mode. The mobile phase for the positive mode was comprised of A: methanol∶acetonitrile∶H2O  and B: isopropanol; both contained 10 mM ammonium acetate and 0.1% (w/v) formic acid. The following gradient was used: 0–5 min, 90% A, 10% B; 25 min, 60% A, 40% B; 60 min, 45% A, 55% B; and 62 min, 45% A, 55% B. The mobile phase for the negative mode was comprised of A: methanol: acetonitrile: water  and B: isopropanol. Both A and B contained 0.025% (w/v) ammonium hydroxide. Samples were eluted with a gradient elution solution as follows: 0 min, 100% A; 10 min, 95% A, 5% B; 12 min 45% A, 55% B; and 30 min 45% A, 55% B. For each sample, the column was re-equilibrated with A for 10 min before gradient elution. The temperature of the columns was maintained at 40°C, and the flow rate was 0.2 mL min−1.For quantitative analysis, the protonated forms , including TAG 17∶0/17∶0/17∶0 , MGDG 18∶0/18∶0 , DGDG 18∶0/18∶0 (Avanti Polar Lipid), PE 14∶1/17∶0 (Avanti Polar Lipid), PG 17∶0/20∶4 (Avanti Polar Lipid), PC 17∶0/20∶4 (Avanti Polar Lipid), and PI 17∶0/20∶4 (Avanti Polar Lipid). Among these, PC 17∶0/20∶4 was used as an ITSD for both PC and DGTS quantification, and PI 17∶0/20∶4 was used for both PI and SQDG quantification. The external standards (ETSD) for calibration included TAG 16∶1/16∶1/16∶1 , TAG 16∶0/18∶1/16∶0 (for TAG C50), TAG 18∶1/16∶0/18∶1 (for TAG C52), and TAG 18∶1/18∶1/18∶1 (for TAG C54) (Sigma-Aldrich). MGDG 16∶3/18∶3 , DGDG 18∶3/18∶3 (Matreya), PE 20∶4/20∶4 (Avanti Polar Lipid), PG 18∶0/18∶1 (Avanti Polar Lipid), PC 18∶1/18∶1 (Avanti Polar Lipid), DGTS 16∶0/16∶0 (Avanti Polar Lipid), and SQDG  were used as ETSDs for the corresponding classes of membrane lipids. ETSD were titrated relative to a constant amount of ITSD to establish the correlation between the ratio of the analyte signal to the ITSD signal and the ratio between their concentrations.p value ≤ 0.05, the differences between two cell forms were interpreted as being significant.Student's t-test was used to compare the cellular content of pigments, lipids, proteins and carbohydrates (n = 6) between given two cell forms, as well as for the semi-quantitative results of immunoblotting (n = 2). If the test gives Under the favorable LL condition, the motile cells were usually pear-shaped with a pair of flagellae at the anterior end, and the protoplast of the motile cell was enclosed by a swollen, gelatinous extracellular matrix Fig. 1A.a and chl b than palmella cells by 17.8% (p<0.01) and 22.8% (p<0.01), respectively. However, the ratio of chl a to chl b in these two cell types was similar. The β-carotene content in both cells was also similar (ca. 2.30 mg g−1 DW). Neither canthaxanthin nor astaxanthin was detected in motile cells, whereas the palmella cells accumulated small but noticeable amounts of these two carotenoids under LL , and from 5.11 to 8.36 in red cysts transformed from palmella cells (RC-P)  and 48.2% (p<0.01), respectively. After 24 h exposure to HL, astaxanthin in RC-M equaled 4.96 mg g−1 DW, which was 64.7% greater than in RC-P (p<0.01).After exposure to HL for 24 h, chl s (RC-P) Table 1.max was lower than that of palmella cells. The saturation irradiance for palmella cells is 323 µmol m−2 s−1, significantly higher than that for motile cells (231 µmol m−2 s−1). These results indicated that palmella cells may possess more pronounced capacities for high light adaptation, but motile cells can dominate in the environment with lower irradiance. The initial slope of RC-M and RC-P was slightly declined as compared to motile cells and palmella cells, respectively. For both RC-M and RC-P, ETR didn't reach a saturation level under the highest light intensity (849 µmol photons m−2 s−1) tested.To investigate the changes in photosynthetic capacity during encystment and HL acclimation, the photosynthetic efficiency of PS II of different cell forms were measured by using chlorophyll fluorometry under varying light intensities Fig. 2. −2s−1 . Under the strongest irradiance (849 µmol photons m−2 s−1), no significant difference with respect to Y(II) was observed for all the cell forms.Motile cells exhibited higher Y(II) than palmella cells under light intensities of 30–555 µmol photons m−2s−1 Fig. 2B,1Chl to the ground state by the emission of excess energy as heat. This process is affected by acidification of the thylakoid lumen and the xanthophyll cycle, and is thus considered a regulatory mechanism protecting algae and higher plants from excess light −2 s−1  during encystment under LL (p<0.05) and 35% (p<0.05), respectively. In contrast to the attenuation of PSII and cytochrome b6f, PsaA, a core reaction center protein of PSI, was 34% higher in palmella cells than in motile cells (p<0.05).Several components of the photosynthetic apparatuses in motile and palmella cells were quantified by western blotting Fig. 3A.p<0.05) and 62% (p<0.01), respectively, greater decreases than those seen in palmella cells during HL acclimation  (p<0.05), whereas remained unchanged in motile cells.After exposure to HL for 24 h, D1 protein, PsbO, and PetC decreased in both motile and palmella cells to different extents Fig. 3A.−1 DW protein, 0.221 g g−1 DW carbohydrate, and 0.162 g g−1 DW glycerolipid. The high protein content of the motile cells is in line with their high photosynthetic growth potential. The protein content in palmella cells was lower than in motile cells, and it decreased 36.7% (p<0.01) during the encystment process under LL. During encystment, carbohydrate content increased 27.5% (p<0.01), whereas glycerolipid content decreased 17.9%  and 31.2% (p<0.01) in motile and palmella cells, respectively (p<0.01) relative to the motile cells, whereas little change in carbohydrate content was observed in RC-P as compared to palmella cells. These results indicated that photosynthates were preferentially partitioned into storage carbohydrates (probably in the form of starch) in motile cells under HL.After exposure to HL for 24 h, total protein content decreased 52.8%  were identified in all four types of H. pluvialis cells, i.e., motile cells, palmella cells, RC-M, and RC-P. The galactolipid MGDG was the most abundant membrane glycerolipid in motile and palmella cells grown under the favorable LL conditions, and the glycerolipid content in motile cells comprised up to 72.9 µmol g−1 DW, which was 42.5% greater than in palmella cells  (A lipidomics method was employed to quantitatively measure molecular species of glycerolipids in the different forms of  p<0.01) Fig. 5. Haematococcus cells. PG, the only bulk phosphoglycerolipid found in thylakoid membranes −1 DW, accounting for 6% of total glycerolipids). During the encystment process under LL, PC increased 19.4% in palmella cells  compared to motile cells (5.25 µmol g−1 DW), whereas PI decreased from 7.05 µmol g−1 DW to 5.51 µmol g−1 DW during encystment.Four classes of phospholipids  were detected in p<0.01), 14.7% (p<0.05), 22.7% (p<0.01), and 56.4% (p<0.01), respectively, suggesting a dramatic degradation of the thylakoid membranes and photosynthetic complexes (p<0.01). In motile cells, PE and DGTS were reduced 47.9% (p<0.01) and 22.1% (p<0.05), respectively; however, both remained unchanged in palmella cells after 24 h under HL. In contrast to the remarkable reduction in chloroplast membrane lipids and nitrogen-containing glycerolipids , PI content increased 17.7% (p<0.01) in palmella cells during HL acclimation, and remained unchanged in motile cells. Among the eight membrane glycerolipid classes, PC was the only lipid that increased under HL, which occurred in motile cells .Under HL, MGDG, DGDG, SQDG, and PG content in motile cells decreased 63.6% . Under HL, a number of chloroplast membrane lipids, including MGDG , SQDG (34∶3), and PG (34∶3 and 34∶2), were reduced dramatically in motile cells but remained relatively stable in palmella cells , corresponding to 0.7% of total glycerolipids. During encystment under LL, TAG content increased ca. 7-fold in palmella cells . Accumulation of TAG under favorable culture conditions has recently been reported in Chlamydomonas and Nannochloropsis as well Although TAG synthesis is considered a protective strategy by which microalgae cope with environmental stress  H. pluvialis cells were subjected to HL, the most noticeable change with respect to lipid composition was the accumulation of large amounts of TAG. TAG content in motile and palmella cells reached 70.04 and 43.41 µmol g−1 DW, respectively, after 24 h under HL, which accounted for 36.9% and 25.4% of total glycerolipids, respectively , the motile cells lost their flagellae and became palmella cells with thickened cell walls. Our previous study When The chlorophyll fluorometric analysis demonstrated that the capacity to dissipate excessive excited energy via the NPQ mechanism developed during encystment and was further augmented when palmella cells were subjected to HL. NPQ is a rapid and effective process that is induced seconds after photosynthetic cells are exposed to excess light H. pluvialis cells changed the energy balance between PSII and PSI under HL by enhancing the quantum yield of PSI [Y(I)] while reducing Y(II) b6f during encystment, and increasing cyclic electron transport around PSI are a suite of photoprotective mechanisms developed in palmella cells for acclimation under HL.The adjustment of relative numbers of PSII and PSI complexes represents an important mechanism by which plants and algae prevent photodamage during high-light acclimation H. pluvialis glycerolipids in response to HL and under encystment. The ability of living cells to survive under extreme environmental conditions may rely on their ability to modify their membrane composition and adjust their lipid desaturation level H. pluvialis, which can in turn provide protection from excess light This study revealed for the first time the global remodeling of H. pluvialis cells exposed to higher irradiance accumulated more astaxanthin but exhibited higher cell mortality Although production of ROS and cell mortality were not directly measured in this study, multiple lines of evidence suggest that the motile cells suffered more severe photo-oxidative stress than palmella cells when exposed to HL. First, more profound decreases in the quantum yields of PSII, D1 protein, and PsbO, as well as in several chloroplast membrane lipids  occurred in motile cells than in palmella cells under HL. In oxygenic photosynthetic organisms, PSII and PSI are two major sites of ROS production Diacylglycerol-based polar lipids are the building blocks of the cellular membranes of living organisms. It is generally believed that glycolipids  and the phospholipid PG are the major components of chloroplast thylakoid membranes, whereas phospholipids like PE, PC, PI, and the nonphosphorus betaine lipid DGTS reside in the extraplastidic membranes of photosynthetic cells H. pluvialis. PG showed the most profound decrease among all the chloroplast membrane lipids in both motile and palmella cells; MGDG was dramatically reduced in motile cells and red cysts under HL; by contrast, DGDG and SQDG showed moderate decreases in both motile and palmella cells under the same conditions. The different responses of these lipids may result from their uneven distributions among the photosynthetic complexes and their distinct functional roles in maintaining the proper structure and function of chloroplast membranes.Our results indicate that the major classes of chloroplast membrane lipids exhibit different fates under HL stress in b6f, and DGDG is found in PSII, PSI and LHCII H. pluvialis cells is likely linked to the breakdown of PSII and cytochrome b6f in response to HL, especially in motile cells. In the bilayer chloroplast membranes of Arabidopsis, MGDG can be converted to DGDG to prevent the formation of hexagonal structures and consequent membrane infusion under freezing stress H. pluvialisunder HL may be attributable in part to the conversion of MGDG to DGDG.MGDG and DGDG are two major galactolipids that constitute the bilayer of thylakoid membranes. Moreover, MGDG has been identified in cyanobacterial PSII, PSI, and cytochrome Thermosynechococcus elongates and spinach (Spinacia oleracea) b6f complexes Arabidopsis thylakoid membranes under Pi starvation H. pluvialis under HL.PGs are anionic lipids that are present primarily in PSII, PSI, and LHCII in H. pluvialis mass culture. This phenomenon often occurs in Haematococcus cells at the exponential growth phase, when the cell population is mainly in the flagellate form. Palmella cells can develop a suite of protective mechanisms during encystment to bestow greater resistance to HL than motile cells, and thus, applying palmella cells instead of motile cells to the stressful red stage of cultivation may represent a promising strategy for increasing growth and astaxanthin production. During the first day under HL, astaxanthin productivity (g L−1 day−1) in palmella cells was less than in motile cells. From a biotechnical perspective, high astaxanthin contents are desirable, although higher yields can be achieved be extending culturing time. Astaxanthin accumulation in palmella cells below the maximum potential may be attributable to the fact that palmella cells favor PSI cyclic electron transport over linear electron transport. Unlike linear electron transport, which produces both ATP and NADPH, cyclic electron transport is involved only in ATP production H. pluvialis cells, 90% of astaxanthin is attached with one or two fatty acids Cell death during the first 1–2 days under photo-oxidative stress represents a major loss of biomass in Therefore, our future efforts will explore how to enhance NADPH production in palmella cells as well as how to increase the levels of astaxanthin biosynthetic enzymes through physical or genetic manipulations. Additionally, we will investigate biotic and abiotic factors that stimulate the development of protective mechanisms in palmella cells.


Arg-1/iNOS and CCL22/CXCL10) and the aqueous humors of nAMD patients (CCL22/CXCL10) than in the controls. Our results suggested that the dynamic patterns of M1 and M2 were different in both the experimental and clinical CNV. The M2 macrophages were predominant and may play a more important role in the development of CNV.Macrophages play an important role in the development of age-related macular degeneration (AMD). In this study, the spatial and temporal changes and the polarization of macrophages in murine laser-induced choroidal neovascularization (CNV) were investigated, and the polarized M1 and M2 biomarkers in the aqueous humors of neovascular AMD (nAMD) patients were studied. Macrophages, the main infiltrating inflammatory cells in CNV lesions, were evidenced by a significant increase in F4/80 mRNA expression and by the infiltration of F4/80+ cells in the lesions and the vicinity of laser-induced CNV. The mRNA expressions of M1-related markers were dramatically upregulated in the early stage, while the M2-related markers were slightly upregulated in the middle stage and sustained until the late stage. The results of immunostaining showed a similar early-but-transient M1 pattern and a delayed-but-sustained M2 pattern in laser-induced CNV. In addition, a higher M2/M1 ratio was found in both the murine models ( Advanced AMD is generally classified into two categories: geographic atrophy (aAMD) or ‘dry’ AMD, and neovascular (nAMD) or ‘wet’ AMD. nAMD leads to irreversible vision loss in 90% of patients through neovascularization from the choroid, secondary subretinal hemorrhage, photoreceptor cell death, and ultimately, irreversible fibrovascular scarring. This process is characterized by the proliferation and/or infiltration of various types of cells, including retinal pigment epithelium (RPE) cells, glial cells, fibroblasts and macrophages, which interact with inflammatory cytokines and growth factorsMacrophages have been proved to be able to be polarized, and have been characterized based on their functions as surface markers and their cytokine/chemokine profiles. Classically activated macrophages (M1) and alternatively activated macrophages (M2) are at the opposite ends of this spectrum. M1 macrophages, driven by Th1 cytokines, are generally proinflammatory and secrete M1 chemokines, such as CXCL10 and CXCL11 (chemotactic for activated T cells). In contrast, M2 macrophages, driven by Th2 cytokines, facilitate tissue repair and neovascularization, and secrete M2 chemokines, such as CCL2 and CCL22 (chemoattractants for Th2 cells that enhance fibrosis). However, due to their plasticity, macrophages with one phenotype can easily convert into another phenotype when placed in a new microenvironment. Therefore, identifying the temporal and spatial changes of macrophages during the early and late stages of nAMD can lead to a better understanding of its pathogenesis. However, to date, the phenotype, number, and distribution of macrophages in nAMD remains unknown. In this study, we investigated the spatial and temporal changes of macrophages and the polarized M1 and M2 markers in laser-induced nAMD-like CNV in mice, and in the aqueous humors of human nAMD patients.et al.Laser-induced CNV in mice is currently the most established and commonly utilized model for studying the pathogenesis of CNV. Like the results of Lambert P < 0.05 at D1, D3, D7, and D14; P < 0.01 at D21; Dunnett’s test)  macrophages were found as early as day 1 (D1) after the laser treatment . Of note’s test) . AdditioiNOS, TNF-α, IL-6, CXCL10, and CXCL11, in the mouse eyes were measured before (shown as D0) and 1, 3, 7, 14, and 21 days after the laser treatment (P < 0.05), but were decreased at D7 (P < 0.01). Thereafter, the mRNA expression diminished rapidly (e.g. iNOS) or decreased slowly   . Further CXCL11) . The res CXCL11) . However CXCL11) . However CXCL11) . GeneralArg-1, CD163, CD206, CCL2, and CCL22, were also measured in this study . They gradually increased on D3, significantly peaked at D7, plateaued between D7 to D14, and were sustained to D21 (P < 0.05). Moreover, the mRNA expression of CCL2 was slightly upregulated and plateaued between D1 to D7 , and increased gradually from D14 to D21(P < 0.01). Interestingly, the mRNA expression of Arg-1 dramatically upregulated on D1, and significantly peaked at D3, then decreased at D7 (P < 0.05) and plateaued between D14 to D21  the ratio of Arg-1 (an enzyme highly expressed in M2 macrophages) and iNOS (which competes with Arg-1 for a common substrate and is highly expressed in M1 macrophages), and 2) the ratio of CCL22 (a protein secreted by M2) and CXCL10 (a protein secreted by M1). Ratio values of >1 indicated M2 predominance, and ratio values of <1 indicated M1 predominance10Arg-1/iNOS mRNA ratios were 1.00 (D0), 2.00 ± 0.19 (D1), 4.82 ± 0.46 (D1) (D3), 4.89 ± 1.20 (D7), 4.34 ± 0.94 (D14), and 4.16 ± 1.10 (D21) , 1.33 ± 0.12 (D1), 1.66 ± 0.68 (D3), 5.90 ± 0.24 (D7), 6.10 ± 0.90 (D14), and 6.70 ± 0.76 (D21)  levels and a high M2/M1 chemokine ratio in the retina/RPE/choroid/sclera complex, compared to tissues from normal control mouse eyes. These results indicated that M2 macrophages, rather than M1 macrophages, likely play a more important role in the development of L-CNV in this model, especially in the late stage.The results showed that during the early and late stage of CNV, the increased folding of Arg-1 mRNA is higher than that of iNOS mRNA , and remP < 0.001) was significantly higher than that at other time-points: 1.46 ± 0.17% at D3, 0.65 ± 0.13% at D7, 0.54 ± 0.18% at D14, and 0.50 ± 0.16% at D21 (P < 0.001) (P < 0.05), and peaked at D7 . The results for Arg-1/iNOS showed the same pattern, which was 2.08 ± 0.68 (D0), 0.31 ± 0.05 (D1), 1.10 ± 0.48 (D3), 5.02 ± 1.28 (D7), 4.30 ± 1.67 (D14), and 1.76 ± 1.10 (D21) (P < 0.05)  , was inc < 0.05) . Taken tP value of 0.758. The results showed that the median CCL22 was 157.03 pg/ml (25% to 75%:130.51 to 185.91) in the nAMD patients and 88.41 pg/ml (31.92 to 101.97) in controls (P = 0.037)   .CXCL10 wue 0.004 . In the = 0.037) .P = 0.002) (P = 0.012) (P = 0.008) (P = 0.019) and the maximal neurosensory thickness (MNT) , but was not correlated with the presence of subretinal fluid (SRF), subretinal hemorrhage (SRH), hyperreflective foci (HF), or pigment epithelium detachment (PED), with P values of 0.093, 0.562, 0.174, and 0.320, respectively in the recurrent group, while is 132.95 pg/ml (96.71 to 157.78) in treatment-naïve eyes (= 0.002) . However= 0.012) . A furth= 0.008) . The meapectively. Thus, tet al. found the strong expression of cell adhesion molecules by inner retinal blood vessels, which suggested that the retinal vasculature was activated, facilitating macrophage penetration into the retina through the activated inner blood-retina barrier9Although laser-induced CNV is not a true model of nAMD, its condition in animals follows an acute wound-healing tempo rather than the more insidious pathologic changes and low-grade changes to immune responses observed in nAMD, so it is therefore used widely to unravel the mechanisms of CNV913141517182202122et al.From the data in our study now, it is difficult to define the origin of these macrophages and to differentiate between the proliferation of resident microglia and the recruitment of hematogenous macrophages. However, Liu et al. suggested that M2 macrophages are protective in the aging retina and choroid, moreover, they suggested a tendency toward greater M1 activity in atrophy AMD and greater M2 activity in nAMD, which is basically consistent with our data11Macrophage polarization is highly plastic, depending on the tissue microenvironment, and has been reported in ocular diseases, such as choroidal melanoma and sympathetic ophthalmia262122Arg-1, were slightly upregulated on D1, then gradually increased, peaking on D7 to D14. Furthermore, cell membrane markers of M2 macrophages, CD163 and CD206, and chemokine CCL22 were noted to be upregulated slightly but continuously from D1, and did not peak until D14. The expression of chemokine CCL22, which is produced by activated M2 macrophages, was found to be increased on D7 and to maintain a relatively high level at least until D21. These data suggest that a shift toward the M2 phenotype occurred after the transient shift to M1, and kept the same tempo in the CNV development. Furthermore, in the current study, CCL22 was found to be significantly increased in the aqueous humor of nAMD patients compared to the controls, and was even higher in patients with recurrent nAMD lesions  on D1, we suggest that M1 macrophages are primarily associated with lesions observed in the outer retina in the early stage, and they may be the main effectors of the inflammatory response. Meanwhile, M2 macrophages are tightly associated with the middle and advanced stages, and are coincidental with the development of CNV. Taken together, M2 macrophages, rather than M1 macrophages, are present in a greater proportion in CNV.In order to determine the specific pattern and activation status of these macrophages, we performed intracellular stains for iNOS, which identified M1 macrophages and CD206 production, a hallmark of M2 macrophage differentiation. We observed iNOS + cells within the inner layer of the retina at D1, but CD206 staining was negative, indicating the presence of activated M1 macrophages in the inner layer of retina  and 4. COverall, our work confirms the critical role of macrophages in the development of CNV. We suggest that the dynamic patterns of M1 and M2 in laser-induced CNV are different from each other: an early shift towards M1 immediately, then a delayed-but-sustained shift toward M2. Compared with M1 macrophages, M2 macrophages are predominant and might have a more important effect on the development of CNV. Further investigation is required to understand the mechanisms of macrophage polarization in the pathogenesis of AMD, and how they interact with additional important factors involved in nAMD, such as oxidative injury, complement activation, cell death, and angiogenesis. Targeting these cells and their signaling pathways may be the key to discovering new avenues for the treatment of this disease.Pathogen-free C57BL/6 male mice, aged from 6 to 8 weeks old, were used in this study. All procedures and animal care were performed in accordance with the guidelines of the Association for Research in Vision and Ophthalmology on the use of animals in research, and were approved by the Animal Ethical Committee at Zhongshan Ophthalmic Center, Sun Yat-sen University (Permit Number 2013–012).CNV was induced by laser photocoagulation with rupture of Bruch’s membrane as previously described31To quantitatively analyze the mRNA expressions in eye tissues, real-time RT-PCR assays were performed. Mice at different time-points before and after laser  were prepared for analysis of the expressions of F4/80 (marker of MΦ), Il-6, Cxcl10, Cxcl11,Tnf-α,iNos (marker of M1) and Ccl2,Ccl22,Cd163,Cd206,Arg-1(marker of M2) and beta-actin (housekeeping gene). Briefly, mice were subjected to laser-induced rupture of Bruch’s membrane with 10 laser spots per eye. Twelve age-matched mice without laser treatment were served as controls. Mice were euthanized and eyes were enucleated. The anterior segments were removed after an annular incision along the equator, and the RNA was isolated from posterior pole RPE/choroid/sclera fractions using Trizol reagent  in accordance with the manufacturer’s instructions. Four eye cups from four mice were considered as one sample (12 mice per group). 1 μg of each sample of total RNA, pretreated with DNase I(Promega), was reverse-transcribed into complementary DNA(cDNA) using SuperScript III First-Strand Synthesis System. Each PCR was carried out in a 20 uL volume using SYBR Green I Master (Roche) for 10 minutes at 95 °C denaturation, followed by 40 cycles at 95 °C for 30 seconds and 60 °C for 1 minute in Roche480. To determine whether the expression of the potential internal control gene varies under the experimental conditions, the real time PCR data was presented from the replicate cDNAs as 22Ct. The Delta-Delta-Ct (ddCt) method was used for relative quantification. Primers used were listed in Immunohistochemistry was performed as previously reportedRPE-choroid tissue lysate was used for Western blotting experiments. Freshly dissected posterior eye poles  were lysed with lysis buffer containing RIPA and phenylmethylsulfonyl fluoride (PMSF). After centrifugation, the supernatant was used for Western blotting. Protein concentration was determined by the Bradford–Lowry method and the samples were stored at −80 °C until used. Protein samples (20 μg) were loaded onto SDS-PAGE gels and then were transferred to a PVDF membrane . After being blocked with 5% nonfat milk for 1 h, the membrane were incubated with the following primary antibodies at 4 °C overnight: monoclonal rabbit anti-mouse iNOS, polycolonal rabbit anti-mouse Arg-1. Equal protein loading was assessed using an monoclonal rabbit anti-β-actin antibody. Protein bands were visualized by incubation with anti-rabbit IgG HRP-linked antibody  and chemiluminescence HRP substrates .Sixteen patients with nAMD who received an intravitreal injection of Ranibizumab  were recruited, and 12 age-matched patients with age-related cataracts who underwent routine phacoemulsification surgery served as controls. The exclusion criteria were: 1) age below 50 years old; 2) history of any other ocular diseases, apart from age-related cataract; 3) any previous intraocular surgery or photodynamic therapy or intravitreal triamcinolone acetonide injection; and 4) previous history of any intravitreal anti-VEGF treatment within the last 6 months in the study eye or within the last 3 months in the fellow eye. All subjects underwent a complete ophthalmic examination, including visual acuity using ETDRS, slit-lamp examination, intraocular pressure measurement, fundus examination, fluorescein angiography, and optical coherence tomography . This study was conducted in accordance with Declaration of Helsinki and was approved by the Institutional Review Board of Zhongshan Ophthalmic Center, Sun Yat-sen University. Informed consent was obtained from all patients and controls.® Human Chemokine Array, RayBiotech, Inc., Norcross, GA).The concentration and ratio of CCL22/CXCL10 were analyzed with the Quantibody Q-Analyzer .Undiluted aqueous humor samples (100–200 μl) were obtained through anterior chamber paracentesis before intravitreal ranibizumab injections in nAMD patients or before cataract surgery in control. In control patients, paracentesis was performed before any conjunctival or intraocular manipulation to avoid breakdown of the blood–aqueous barrier associated with surgical trauma. Samples were snap-frozen and maintained at −80 °C until analysis. To assess the concentration of CCL22 and CXCL10 in aqueous humor, a multiplex ELISA method was used according to the manufacturer’s instructions . All examinations were performed by one well-trained technician, and only images with well-defined retinal layers were selected. All the images were read and analyzed by two independent masked investigators. The presence of intraretinal fluid (IRF), subretinal fluid (SRF), subretinal hemorrhage (SRH), hyperreflective foci (HF) at outer retinal layer, and presence of retinal pigment epithelium detachment (PED) was recorded. Greatest linear diameter (GLD) of lesion, maximal retinal thickness (MRT), defined as the maximal vertical distance between the ILM and the surface of the RPE , maximal neurosensory thickness (MNT), defined as the maximal vertical distance between inner limiting membrane (ILM) and outer surface of the retinal photoreceptors were measured as described in prior reports123435th percentile to 75th percentile). Chemokines concentrations were compared using Mann-Whitney U test in different groups. Correlations between cytokine concentrations and OCT data, including GLD of lesion, MRT and MNT, were calculated by Spearman’s correlation test. Two-tailed test or Mann-Whitney U test were used to compare chemokines between nAMD patients with and without IRF, SRF, SRH, HF and PED, respectively. P < 0.05 was considered statistically significant.The data were processed and analyzed statistically with SPSS . For animal study, the means and SEM were calculated for all parameters determined in the study. Data were analyzed statistically using one-way ANOVA. For human study, the data were denoted by median .