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on the other hand cytoplasmic effectors have the duty of dealing with host cells at a much more intricate level cytoplasmic effectors are active once they reach the plant cytoplasm and tend to target plant defense signaling components effectors from p syringae have been shown to target antipathogenic vesicle trafficking and kinasebased recognition activity of the host a prime defense component 29 some effectors may also transit through the cytoplasm to reach their final destination eg organelles

nuclear effectors are seemingly ultimate weapons in the inventory of pathogens since they are thought to suppress the immune response from upstream nuclear effectors could potentially shut off master switches of the immune machinery or reprogram host transcription to the benefit of pathogens a recent investigation of 49 putative effectors from h arabidopsidis revealed that 33 localized strictly to the nucleus and an additional 33 were nucleocytoplasmic 30 since several effectors tend to migrate toward the nucleus it would be logical to assume that some rproteins act in the nucleus indeed several rproteins such as snc1 n and rps4 were found to localize to the nucleus [31] [32] [33] [34] tobacco tirnblrr rprotein n localizes to the nucleus in the absence of its elicitor the tobacco mosaic virus p50 helicase fragment 32 lending support to a default presence of rproteins in the nucleus to monitor their corresponding effectors rather than being relocalized upon effector binding however snc1 and n nuclear accumulation is reduced at elevated temperatures making their mode of action temperaturedependent 35 it was demonstrated recently that eti is more active at low temperatures 1023 c while pti takes over at higher temperatures 2332 c 36 it has also been shown that bacterial pathogens strive and multiply at higher temperatures but secrete their effectors more actively at lower temperatures 37 38 these observations suggest that the immune system of plants is adapted to pathogen physiology however some pathogens prefer more temperate environments around 18 c for optimal growth 3940

computer software such as nod psort ii and wolf psort can predict the subcellular localization of various proteins but that of very few candidate effectors has been verified experimentally [41] [42] [43] relative to the wealth of those from all plant pathogens a number of plant pathogensecreted effector proteins have been reported to localize in the nucleus but most localization studies have been conducted with gfptagged assays it should be noted that gfp fusion may abrogate proper effector localization either by hiding a sorting signal or by inducing change in the 3d structure of native effectors which could prevent interaction with a protein involved in true effector localization in addition most of these experiments are transient assays and do not examine localization during infection therefore although gfp represents a very powerful tool at our disposal to identify subcellular effector localization care should be taken when analyzing the results however since gfp does not diffuse to the nucleolus it is safe to assume that nucleolar localization is effectordriven rxlr effectors such as harxll3b haatr13 emoy2 and harxl44 from hyaloperonospora arabidopsidis localize to the nucleolus of plant cells 30 in phytophtora capsici crn effectors all localize to the nucleus and at least two have been found to accumulate in the nucleolus suggesting that there might be subnuclear localization domains 44 the nucleolus is a multifunctional subcellular organelle critically involved in ribosome biogenesis and protein synthesis 45 several dna viruses and retroviruses are known to target the nucleolus umbravirus orf3 potato leafroll virus capsid protein and influenza virus nucleoprotein are some examples of viral proteins localizing to the nucleolus [46] [47] [48] [49] given that viruses are entirely dependent on the host machinery to translate their genome into proteins they are expected to target the nucleolus however one can wonder why biotrophic filamentous pathogens would target this subnuclear compartment the effector harxl44 from the obligate biotrophic pathogen h arabidopsidis was recently shown to target nucleolar and nuclear mediator subunit 19a med19a this interaction results in med19a degradation in a proteasomedependent manner med19a degradation appears to shift transcription from salicylic acidresponsive defense to jasmonic acid and ethyleneresponsive transcription thereby conning the host to enhance its susceptibility 50

what happens once a pathogen gets access to its host how does the host respond to the pathogens demands and what are the overall cellular dynamics in play answering such questions becomes a lot more imperative when dealing with obligate biotrophs because of their intimate relationship with the host and since they can only survive in living cells obligate biotrophic pathogens thus have to be subtle when dealing with their host after invasion first of all they have to keep host immunity in check at all times by suppressing pti second they have to continuously feed from plant cells finally they need to steadily propagate and multiply

fungal spores grow on plant surfaces upon germination it has been shown that the rust fungus uromyces appendiculatus uses topographical cues for orientation and the formation of infection structures 51 once u appendiculatus detects a 05m ridge which it interprets as the presence of the stomatal lip its entry point into tissue it starts producing its infection structure 51 when the pathogen has forced its way into plant tissue nutrient acquisition and defense suppression occur primarily through haustoria although effectors are also released from growing hyphae support for such a mechanism is lent by deep sequencing of the biotrophic growth phase of colletotrichum higginsianum during a thaliana infection 52 in this pathosystem effector genes are expressed in consecutive waves associated with pathogenic transition and some are expressed before host invasion at the appressorial stage 52 in fact multistage transcriptome analysis of melampsora laricipopulina the causative agent of the poplar leaf rust obligate biotroph revealed that a number of smallsecreted proteins were even expressed in resting urediniospores 53 therefore we can infer that suppression of plant immunity starts prior to the formation of haustorial structures in host tissue while our understanding of molecular partners at play is progressing we have made few inroads into the establishment of planthaustoria interactions and postinvasion events dynamic interplay could be mainly driven by the invader and as we progress in this review we will examine some important phenomena that may hold clues to these questions

it should not be difficult to conceptualize massive host cellular reprogramming occurring in response to the development of haustoria haustoria are found to be surrounded by endoplasmic reticulum actin cytoskeleton and cytoplasm along with the accumulation of golgi bodies and mitochondria 54 it has also been observed that a significant amount of tonoplast is present around these complexes 54 to host such critical appendages cells have to expand their plasma membrane tremendously haustoria are separated from the host cytoplasm by an extrahaustorial matrix ehm the ehm has been speculated to be mostly of host origin sealed from haustoria by a hautorial neck band 55 56 however it differs from the plasma membrane in both cytological and biochemical properties 55 57 the ehm also appears to vary in composition over time 58 59 recently lu et al 60 reported that some plasma membrane resident proteins relocalize to the extrahaustorial membrane during infection for example the aquaporin pip14 and the calcium atpase aca8 remained at the plasma membrane during infection with either h arabidopsidis or phytophtora infestans while the syntaxin pen1 penetration deficient 1 the synaptotagmin syt1 and the remorin strem13 were present in the extrahaustorial membrane around p infestans haustoria interestingly this relocalization appears to be pathogendependent since prr fls2 localized in the ehm of p infestans but remained at the plasma membrane and was excluded from the ehm in h arabidopsidis however the most remarkable feature of this cellular rearrangement is the position of the nucleus studies have shown that the arabidopsis nucleus stays close to h arabidopsidis haustoria 30 and this is presumably driven by the actin cytoskeleton 61 62 it is possible that proximity of haustoria to the nucleus enables pathogens to deliver their effectors more quickly to the nucleus for cell reprogramming proximity of the nucleus to the intruder would thus be driven by the pathogen per se but one cannot exclude that host plants could steer this process autonomously to respond quickly to pathogen attack

pathogens are known to target host vesicular trafficking a key element of plant defense 30 in h arabidopsidis 26 of examined effectors have been found to localize to membranes the majority of them 18 associating with the endoplasmic reticulum 63 arabidopsis cells hosting h arabidopsidis haustoria develop bulging vesicular structures compared with noninfected cells 30 the occurrence of such vesicles being attributed to presence of the pathogen it is possible that the formation of these vesicles is driven by a particular effector or effectors to upset vesicular movement and disrupt any organized defense response they may also be pathogendriven and provide the extraphospholipid bilayer required at the plasma membrane to accommodate fastexpanding haustoria regardless support for the fact that these are vacuolar structures comes from the observations of very similar structures in cotyledons of transgenic arabidopsis tipgfp plants 64 other types of membrane structures have been shown to differentially localize around haustoria formed by h arabidopsidis and p infestans 60 harxl17 localizes to the ehm during infection by h arabidopsidis however in the absence of the pathogen it localizes to the tonoplast where its ability to enhance plant susceptibility is possibly linked with a task in plant cell membrane trafficking 30 since tonoplast is located close to the ehm along with the effector harxl17 in the event of infection the effector may be interfering with plant cell membrane trafficking and interestingly this also suggests a role for tonoplast in ehm formation however no single effector has been reported to cause the bulblike vesicular structures observed in the presence of growing pathogens 29 and it is not clear whether it is a plant defense response or an effectordriven process surprisingly our understanding of the detailed mechanism of vacuolar biogenesis is still limited justifying the need to push the investigation further into such peculiar vesicular structures it is difficult to elucidate possible pathways being targeted by pathogens to hinder vesicular trafficking and eventually give rise to these bulblike structures in a thaliana a point mutation in the deubiquitinating enzyme amsh3 renders cells incapable of forming central lytic vacuoles in addition amsh3 mutant cells accumulate autophagosomes and incorrectly sort their vacuolar protein cargo 65 vacuoles are important in various plant defense mechanisms and two vacuolemediated mechanisms have been postulated to affect programmed cell death 66 in one of them vacuolarprocessing enzymes mediate vacuolar membrane disruption thus releasing vacuolar content into the cell cytoplasm demonstrated for viral infection 67 in the second proposed mechanism vacuole fusion with the plasma membrane enables the extracellular release of vacuolar content demonstrated in bacterial infection 68 interestingly and coincidentally phenotypic similarity between vesicular structures from amsh3 mutants and cells hosting haustoria can be noticed 60 65 this concurring vesicular signature suggests that pathogens could be targeting amsh3 or similar components to alter the vesicular pathway

octomericexocyst complexes could also be targeted by pathogens given that the exocyst architecture plays an important role in vesicular tethering and redefining cell polarity which are integral to plant defense responses 69 targeted exocytosis occurs during infection and freshlysynthesized defenserelated compounds are delivered to infection foci which eventually leads to asymmetrical plasma membrane development small gtpases from the rab and rho families are known to be essential in this process which involves delivery anchoring and integration of secretory vesicles to the plasma membrane 70 71 whereas the exocyst complex works as a scaffold in tethering operations 72 73 the final process of attachment is mediated by the integral membrane proteins vsnare and tsnare where plasma membrane and vesicle bilayers are fused together to complete the process 74 75 it has already been demonstrated that upon mutating two exocyst subunitsexo70b and exo70h1 from arabidopsis plantsare more susceptible to infection validating their importance in plant immunity 69 pen1 is a classic example of proteins preventing penetration by pathogens pen1 encodes a syntaxin known to interact with the snare proteins snap33 and vamp72 76 and regulates papillae formation in cells under attack 77 papillae are bellshaped cell wall appositions deposited in epidermal cells within papillae various secondary antimicrobial metabolites accumulate along with lytic enzymes and reactive oxygen species which stops the pathogen penetration peg in arabidopsis pen1 is found in significant amounts when the nonhost fungus blumeria graminis f sp hordei endeavors an unsuccessful invasion however when the host fungus erysiphe cichoracearum successfully penetrates arabidopsis cells pen1 is then downregulated 77 the pen1 single mutant allows increased penetration of the nonhost fungus b graminis f sp hordei thereby showing that pen1 helps in procuring an effective penetration barrier 77 thus pen1 could participate actively in polarizing secretion events that lead to papillae formation 77

obligate biotrophic phytopathogens have evolved a robust and elaborate offensive strategy to invade their host by deploying numerous effector proteins it appears that the effectors inventory of pathogens is organized around different types of molecules which have unique capabilities and functions therefore most socalled effectors should be considered candidate effectors a crude way to envision effector deployment is to see apoplastic effectors at the onset of attack performing all the bullwork and setting the stage for more sophisticated weaponry true cytoplasmic effectors could act at the intermediate stage by deactivating local surveillance paving the way for nuclear effectors to enter the nucleus taking over the entire defensive network and stalling the complete immune setup nucleolar effectors from various pathogens are increasingly being reported 44 78 79 and it is likely that they have an important function in pathogenesis many cellular processes including plant defenses depend on the formation of new proteins thus further study needs to be undertaken to understand the task of nucleolar effectors some effectors are also involved in disrupting vesicle trafficking and as such they may be compromising vacuolar integrity which is believed to play a significant role in plant defense plant cells hosting haustoria experience unique cellular rearrangements that are likely influenced by haustoria themselves and driven by secreted effectors

as genomesequencing costs are falling the full sequences of many more genomes are becoming available despite the dazzling speed at which effector catalogs can be assembled functional study of effectors remains a relatively slow and strenuous process in obligate biotrophs functional studies of effectors by virulence assays are hindered by the lack of molecular genetic approaches as a result alternative tactics with heterologous systems are increasingly being adopted given the very large repertoire of effectors observed in obligate biotrophic fungi such as rusts that encode over 1000 small secreted proteins 80 81 one could propose that the outcome of each effector may be a lot more subtle than the bacterial effectors of pseudomonas syringae that have roughly 30 or so effectors 82 and a direct quantifiable impact on virulence may prove difficult to observe since the cumulative result of many effectors may be required alternatively redundancy could explain the huge number of effectors in filamentous pathogens in either case deciphering the interactions of these effectors will likely reveal many unknown components of various plant processes with these issues in mind localization remains one of the first aspects to consider when assessing effector functions in addition combination of genetic evidence and proteinprotein interaction approaches either yeast twohybrid assay coimmunoprecipitation or bimolecular fluorescence complexes may prove to be the best ways of investigating effectors from biotrophic pathogens

no potential conflicts of interest were disclosed" "Chaudhari

Prateek. Ahmed

Bulbul..." Effector biology during biotrophic invasion<br>of plant cells Virulence Not provided. 0 4371

9a8ffb71928a37a550e689f36fa3196493e036fe it has been proposed that cholesterol in host cell membranes plays a pivotal role for cell entry of hiv however it remains largely unknown why virions prefer cholesterolrich heterogeneous membranes to uniformly fluid membranes for membrane fusion using giant plasma membrane vesicles containing cholesterolrich ordered and cholesterolpoor fluid lipid domains we demonstrate that the hiv receptor cd4 is substantially sequestered into ordered domains whereas the coreceptor ccr5 localizes preferentially at ordereddisordered domain boundaries we also show that hiv does not fuse from within ordered regions of the plasma membrane but rather at their boundaries ordereddisordered lipid domain coexistence is not required for hiv attachment but is a prerequisite for successful fusion we propose that hiv virions sense and exploit membrane discontinuities to gain entry into cells this study provides surprising answers to the longstanding question about the roles of cholesterol and ordered lipid domains in cell entry of hiv and perhaps other enveloped viruses "cell membranes consist of numerous proteins and lipids exhibiting complex behavior that includes organization into dynamic nanodomains enriched in sphingolipids and cholesterol that are sometimes referred to as lipid rafts 1 2 accumulating evidence indicates that membrane domain organization plays a vital role in cell signaling and adaptive immune responses to combat infections by many pathogens 3 4 5 for example hiv has evolved to exploit organized membrane domains to gain entry into host cells 6 7 8 9 it is well established that recognition and attachment of hiv to host cells are mediated by the binding of the viral envelope glycoprotein gp120 to the cellsurface receptor cd4 and a coreceptor ccr5 or cxcr4 10 11 after attachment membrane fusion mediated by subunit gp41 of the envelope glycoprotein leads to cell entry because cd4 has been found to associate with detergentresistant fractions of the cell membrane it has been assumed that cholesteroland sphingomyelinrich rafts are platforms for hiv entry 12 13 however because lipids in cholesterolrich regions are more tightly packed and more ordered than those in the surrounding membrane these sites may seem energetically unfavorable for membrane fusion thereby raising doubts about the involvement of ordered lipid regions in the fusion step of hiv entry

because ordered lipid domains are thought to be dynamically fluctuating nanoscopic assemblies of lipids and resident proteins in living cells visualization of the potential involvement of these membrane regions upon entry of viral particles into intact cells remains technically challenging 14 15 in a first step toward solving this problem we showed recently that model membranes with coexisting microscopic ordered and fluid lipid domains were useful in proving that the fusion peptide of gp41 interacts preferentially with ordereddisordered lipid domain boundaries and that these boundaries are also the preferred sites for fusion peptidemediated membrane fusion 16 however these discoveries raised the important question of whether the results obtained in this highly artificial model system could be translated to biological membranes that contain the hiv receptor and coreceptor and to virus particles bearing the full trimeric gp120gp41 complex that is whether membrane domain boundaries would still be the preferred sites for virus attachment andor membrane fusion at the plasma membranes of appropriate target cells therefore we developed a new approach to measuring binding and fusion of hiv particles with plasma membranes to assess the role of membrane heterogeneity in these processes

we used giant plasma membrane vesicles gpmvs derived from hela cells that stably express the cd4 receptor and ccr5 coreceptor cd4 ccr5 investigated the lateral partitioning of both receptors in these membranes and imaged the preferred regions of hiv binding and fusion at the singleparticle level fig 1a gpmvs show temperaturedependent micrometerscale liquidorderedliquiddisordered lold phase separation and have been extremely useful to study the dynamics and lateral distribution of resident membrane proteins 17 18 using this approach we found that lipid order discontinuities in heterogeneous receptorand coreceptorcontaining plasma membranes are important for gp120mediated receptorcoreceptor targeting and gp41mediated membrane fusion

a number of studies have been conducted to localize cd4 and ccr5 in lymphocyte cell membranes although the association of cd4 with lipid rafts is generally accepted divergent conclusions have been reached regarding the raft localization of ccr5 6 12 confirming previous results we observed by immunofluorescence that cd4 colocalized with raftresident ganglioside gm1 in cd4 ccr5 hela cells whereas ccr5 colocalized only partially with gm1 fig s1 when we prepared gpmvs from cd4 ccr5 hela cells by gentle formaldehyde and dithiothreitol dtt treatment movie s1 and fig s2 a and b which forms membrane blebs by releasing otherwise intact cell membranes from the cytoskeleton 17 18 we found that most gpmvs showed microscopic lold phase separation below 25c movie s2 and fig s2 c to e as expected from immunostaining of intact cells cd4 and gm1 were substantially excluded from the ld region in gpmvs indicating the partitioning of cd4 into the lo region fig 1 b and c preferential localization of ccr5 at the boundaries between lo and ld phases occurred and was evident in cellattached blebs and celldetached gpmvs fig 1 b and c this result might explain the debate about the controversial association of ccr5 with lipid rafts ccr5 is not associated with lo or with ld lipid phases but accumulates at the boundaries between them very similar results were obtained with an alternative procedure to induce membrane blebs by the application of small concentrations of nethylmaleimide nem fig s3 suggesting that the results are independent of the blebbing agent and that the resulting gpmvs do not have serious membrane defects

recognition of lold membrane boundaries by hiv next we examined whether and how hiv envelope env particles interact with gpmvs murine leukemia viruses mlvs pseudotyped with hiv gp120gp41 and fluorescently labeled with mkogag were incubated with cd4 ccr5 gpmvs we visualized bound particles by epifluorescence microscopy and found that they migrate along the preparation of largescale phaseseparated gpmvs facilitates the study of the lateral distribution of cd4 and ccr5 and the role of ordered lipid domains in hiv entry lo and ld phases on gpmvs are indicated with red and blue lines respectively b and c partitioning of cd4 and ccr5 in cellattached b or celldetached c gpmvs gpmvs were first stained with 11didodecyl3333tetramethylindodicarbocyanine perchlorate dii at 4c for 60 min and then incubated with fluorescentlabeled ctxb alexa fluor 555 anticd4 alexa fluor 488 or anticcr5 alexa fluor 647 antibodies at 4c for 60 min epifluorescence images of gpmvs performed at room temperature 22c show largescale lold phase coexistence in which dii and ctxb are used as markers of ld and lo phases respectively the selected rectangles are enlarged and shown below d hiv binding to cellattached gpmvs cellattached gpmvs left and viruses labeled with mkogag center are visualized by brightfield and epifluorescence microscopy respectively image overlay right shows that some bright dots hiv env particles indicated by arrows are sitting on the gpmv surface top time series of images from the selected box region shows that the particles bound to gpmv are free to move laterally along the gpmv surface bottom a movie version of this figure is available movie s3 e and f hiv binding to the boundaries between lo and ld phases in cellattached e or celldetached f gpmvs gpmvs left and hiv env particles center were labeled with dio and mkogag respectively g quantification of virions bound to three different regions lo ld and lold boundary of the gpmvs data are means sd of triplicates a total of 120 virions were analyzed for their distribution between the three compartments the largest fraction of virions was found in lold boundary regions scale bars 10 mm the gpmv surfaces fig 1d and movie s3 staining the gpmvs with the ld marker 33dilinoleyloxacarbocyanine perchlorate dio showed that most bound particles localize to lold boundary regions in cellattached fig 1e movie s4 and fig s4 and celldetached gpmvs fig 1f and movie s5 we quantified the virions bound to three different regions lo ld and lold boundaries as shown in fig 1g to assess whether the targeting of the virions to the lold interface is specific to hiv env we also performed similar experiments with mlvs pseudotyped with the env g protein glycoprotein from vesicular stomatitis virus vsvg vsvg virions preferentially bound to the ld phase in gpmvs fig s5 indicating that the lold boundary interaction is not a general feature of all viral particles and requires hiv env given that cd4 is initially localized in lo phase regions of the membrane it seems reasonable to propose that hiv virions bind to cd4rich lo phase membrane regions and then move to domain boundaries where they also engage with ccr5 ccr5 binding and the preferential targeting of the gp41 fusion peptide to lipid phase boundaries 16 likely combine to direct the particles for fusion at these boundaries

to determine whether hiv env particles indeed fuse with gpmvs at lipid phase boundaries we first examined whether they fuse with gpmvs in a bulk lipid mixing assay virions were labeled with selfquenching concentrations of the fluorescent lipid probe r18 and mixed with unlabeled gpmvs lipid mixing was only observed when the gpmvs contained both cd4 and ccr5 but was negligible with gpmvs that were derived from hela cells lacking receptors and coreceptors fig 2a demonstrating the specificity of the hivgpmv fusion system we found that lipid mixing is strongly suppressed by the wellknown hiv entry inhibitors maraviroc and enfuvirtide fig 2b maraviroc inhibits binding to the coreceptor ccr5 19 and enfuvirtide inhibits fusion by blocking the required conformational change of gp41 20 maraviroc reduced the targeting of hiv virions to phase boundaries fig 2c further supporting the notion that ccr5 recognition occurs at these boundaries by contrast enfuvirtide had little effect on the boundary preference of hiv binding while still blocking fusion fig 2d suggesting that boundary binding alone is not sufficient for fusion it requires the energy from refolding gp41 into the sixhelix bundle structure as well

we also observed fusion with gpmvs at the singleparticle level the fluorescence of many hiv env particles that were bound at lold phase boundaries spread over time indicating that the particles fused with the gpmvs fig 2e in addition we carried out electron cryomicroscopy cryoem in an attempt to directly visualize the process of fusion of viral particles with gpmvs as previously observed for bare mlvs containing only gag and gagpol 21 the spherical or tennis racketshaped particles showed a characteristic morphology with a capsid surrounded by the viral envelope fig 2f representative electron micrographs obtained from four different incubations with two separate viral and two separate gpmv preparations show hiv env particles in intimate contact with the gpmv surfaces fig 2g and fig s6 we also observed membrane perturbations in very confined areas of the gpmv lipid bilayer that may represent fusion intermediates fig 2g to ensure that the observed contacts were specific controls with gpmvs lacking cd4 and ccr5 were mixed with hiv env particles fifteen to 30 randomly selected gpmvs per grid were imaged for three different samples and no virusgpmv fusion events were observed however the present resolution of the cryoem images does not permit a distinction between lo and ld phases and is thus not sufficient to determine whether fusion occurs at the lold phase boundaries

next we examined whether the lold phase coexistence on gpmvs is required for hiv env particle binding andor fusion to disrupt lo phase domains we treated gpmvs with methylbcyclodextrin mbcd which depletes cholesterol from the membranes mbcd treatment of gpmvs led to a marked change from approximately circular lo domains to corrugated shapes that are characteristic for bilayers in the gel phase fig 3a and movie s6 however cholesterol extraction did not significantly alter the overall surface expression levels of cd4 or ccr5 on gpmvs fig 3b virion binding was quantified by directly counting the number of particles attached to gpmvs fig 3c or by a centrifugationbased assay fig s7 regardless of the method particle binding was high and not diminished by cholesterol depletion of the gpmvs indicating that the presence of lo domains is not required for the binding of virions however hiv env particles did not bind to gpmvs lacking cd4 and ccr5 further confirming that their attachment is mediated by these receptors rather than lipid phase separation

contrary to virion attachment disruption of the lo phase domains in gpmvs by mbcd significantly decreased the efficiency of fusion of hiv env virions with gpmvs fig 3d this result agrees with a previous report demonstrating a decrease of hiv cell entry after depletion of cholesterol from the host cell membrane 22 the inhibitory effect on fusion by the disruption of lo phases and lold phase boundaries in gpmvs suggests that the unique properties of these boundaries are responsible for attracting receptors and coreceptors and the high fusogenicity of hiv env virions at these sites thus the same physical principles that promote fusion peptidemediated fusion in lipid model membranes namely line tension and lipid packing defects at lold phase boundaries 23 also provide a significant driving force for membrane fusion of hiv env viral particles with biological membranes

lysolipids which adopt an invertedcone molecular shape have been reported to inhibit fusion in a wide variety of systems owing to their propensity to induce positive intrinsic membrane curvature 24 25 here we tested whether lysolipids affect the lipid phase behavior of gpmvs and the ability of hiv to fuse with them lysosphingomyelin lysosm partially dissolved the lo domains of gpmvs into much smaller domains whereas lysophosphatidylcholine lysopc had little effect on lo domains fig 4a and movie s7 although both lysosm and lysopc share a choline headgroup and a similar molecular shape they may differently affect line tension at phase boundaries lysosm is a linactant that appears to reduce line tension whereas lysopc probably partitions more uniformly into the ld phase with a lesser effect on line tension hiv env virion binding was not affected by lysosm or lysopc fig 4b however lysosm significantly reduced the fusion efficiency of hiv particles whereas lysopc had only a moderate effect fig 4b together these results again support the notion that the site of fusion is the domain boundary region where inhibitory lysosm is enriched but lysopc is not enriched the same effect could be triggered enzymatically treatment of gpmvs with phospholipase a2 pla2 which hydrolyzes glycerophospholipids including pc to lysopc had little effect on the lipid phase behavior of gpmvs and hiv fusion whereas sphingolipid ceramide ndeacylase scdase which converts sm into lysosm dispersed the lo domains which eventually spread over the entire gpmv surface and significantly inhibited fusion fig 4 c and d note that the fluorescent lipid probe dii always marks the ld phases in these images the apparent reversal of contrast in some images is due to different levels of cholesterol that can change the connectivity of lo phases as previously demonstrated in model systems 26

to further probe the influence of lysolipids on lipid phases and hiv fusion in welldefined model membranes we used giant unilamellar vesicles guvs and large unilamellar vesicles luvs composed of bsmbpcbpscholesterol 2111 as models 16 only lysosm significantly changed the lo domains in guvs fig 4e and inhibited the fusion of luvs with hiv fusion peptidedecorated virosomes whereas lysopc had only a small effect at the highest concentration fig 4f the domain edges first undulated before larger invaginations formed eventually dispersing the domains into much smaller ones movie s8 regulating the size of lipid rafts by using lysosphingolipids has significant consequences not only on hiv entry but also on cell signaling for example it is well known that sphingosine1phosphate is a potent mediator of numerous signaling pathways 27 and the finding that these signaling mechanisms could be potentiated by raft regulation may be an interesting topic for future investigations as previously reported the recruitment of some proteins such as ras and rac1 to domain boundaries may be more general and may contribute to the regulation of signaling pathways by regulating the nature and size of lipid rafts in these systems 28 29

supported lipid bilayers generated from synthetic and natural lipids have been widely applied since their original introduction 30 including more recently to study fusion of single particles 16 31 the planar geometry of supported membranes has the advantage over guvs that a large extended membrane can be observed by total internal reflection fluorescence tirf microscopy and therefore that large numbers of single events can be simultaneously recorded with high signaltonoise ratio in each experiment leading to greatly improved statistics 32 to exploit this advantage for biological membranes as well we have developed supported planar plasma membranes sppms for singleparticle fusion studies fig 5a our strategy for preparing sppms is to fuse gpmvs onto polymersupported lipid monolayers that have been transferred from a langmuir trough 33 which is different from previous methods 34 and preferentially orients membrane receptors with their binding sites accessible from solution similar to plain supported lipid bilayers 26 35 sppms prepared in this fashion display micrometersized domains in which raft and nonraft lipid components are laterally mobile fig s8 in agreement with the observations with gpmvs the overlay of cd4 and ccr5 images in sppms shows that cd4 was substantially associated with lo domains whereas ccr5 was preferentially located at the edges of the domains fig 5b and fig s9 we observed by tirf microscopy that single hiv env particles bound predominantly to domain boundaries whereas most vsvg particles bound to ld phases on sppms fig 5 c and d in addition these experiments recapitulate the observation made with gpmvs that the location of hiv binding to sppms is determined primarily by the location of the receptor and coreceptor in these membranes and not by the lipid phases fig 5e

we observed large numbers of singlemembrane fusion events of hiv env particles with sppms fig 5f and movie s9 and classified them as docking hemifusion or full fusion fig 5g in lo ld and lold compartments of the sppm we observed no direct full fusion and very little hemifusion in ld phases and only 11 3 direct full fusion and 13 4 hemifusion of hiv virions in the lo phase domains fig 5h in contrast hiv virions fused very efficiently 56 3 of all particles at the lold domain boundaries and an additional 9 4 hemifused in these locations fig 5h these results are qualitatively similar to but quantitatively more robust than those obtained in gpmvs and therefore validate sppms as a useful additional system to quantitatively analyze fusion in heterogeneous biological membranes at the singleparticle level

membrane fusion in hiv entry is a highly cooperative process that must overcome several energy barriers associated with different fusion intermediates this energy must be provided and released at the right time and at the right place to drive the merger between the viral and target membranes although cholesterolrich lipid domains have been implicated in cell entry their proposed involvement was puzzling because ordered lipids are thought to be detrimental to membrane bending and fusion however we recently discovered that lold phase boundaries facilitate fusion in model systems and demonstrated that line tension at these boundaries might provide an additional previously unrecognized driving force for fusion 23 this previous work was limited because no receptors or coreceptors were present and because it could be argued that pure lipid model membranes oversimplify biological membranes to overcome these limitations we demonstrated in this work that a very similar mechanism governs hiv fusion with biological host membranes containing its natural receptors we directly observed that lipid domain boundaries are the portals for hiv binding and membrane fusion presumably because they present the weakest points in the host membrane on the basis of these results we propose the following model hiv gp120 binds to cd4 receptors located in cholesterolrich lipid domains which exist transiently in the plasma membranes of living cells if located near ccr5 coreceptors at domain boundaries this initial binding leads to structural changes of gp120 and binding to the ccr5 coreceptor enriching bound virions at the boundaries fig 6a after cd4 and ccr5 binding gp41 changes its conformation to expose the fusion peptide to lipids in the boundary region 16 the predisposed preference of ccr5 for membrane domain boundaries greatly facilitates the targeting of the fusion peptide to these same fusionpromoting areas of the host membrane although we did not examine the other hiv coreceptor cxcr4 in this study it might function equivalently to ccr5 in recruiting virions to domain boundary regions for more efficient fusion because receptors cooperate with membrane domain boundaries to form hiv entry sites it would be interesting to examine in future studies the binding and fusion efficiencies of hiv particles with gpmvs or sppms where cd4 and ccr5 are distributed differently or randomly

in contrast to many other enveloped viruses hiv displays only a few 14 trimeric glycoprotein spikes on its envelope 36 this small number is certainly inefficient for hiv entry and fusion however the cooperativity of hiv recognition and fusion at membrane domain boundaries may be a unique way to overcome this deficiency and may provide enough energy to drive fusion through its transition states with low env copy numbers just as in the model systems 23 line tension at the boundary likely serves as an additional driving force for fusion it is well known that cd4 t cells play a central role in the immune response following t cell antigen receptor engagement which triggers t cell activation and proliferation against invading pathogens 37 38 t cell activation elicits the recruitment of a number of proteins to ordered lipid regions 39 and results in the formation of larger membrane domains which can be exploited by hiv to facilitate entry fig 6 b and c hiv exhibits infection at a much lower frequency in nave cd4 t cells than in activated cd4 t cells 40 41 suggesting that hiv infection may be associated with the coalescence of cholesterolrich lipid domains just as in model systems we have shown here that line tension may also be a dominant force that promotes membrane fusion in biological membranes the boundaries of the larger lipid domains in activated t cells could contribute to the elevated fusogenic capacity of activated t cells versus resting t cells it is possible that activation of the immune system against other invading pathogens makes the cells more prone to hiv entry through the described mechanism therefore enhanced domain activation could lead to an elevated production of hiv in people infected by other pathogens and thus an acceleration of the progression of aids fig 6d

finally this work also showed that gpmvs and a novel procedure to produce supported planar gpmvderived membranes that we call sppms are very useful to study the selected targeting and membrane fusion of hiv virions we anticipate that these methods will also be useful to study the binding fusion and properties of many other particles on cellderived plasma membranes the new approaches are versatile and should lead to many new discoveries regarding the role of lipid and protein heterogeneity of cell membranes they may also serve as novel platforms for screening of viral entry inhibitors

most lipids and fluorescent probes were from avanti polar lipids and dio dii did and octadecyl rhodamine b chloride r18 were from invitrogen mbcd nem and fluorescein isothiocyanate alexa 647 or alexa 555labeled ctxb were purchased from sigmaaldrich formaldehyde and dtt were purchased from avantor and rpi respectively alexa 488labeled cd4 antibody and alexa 647labeled ccr5 antibody were purchased from novus biologicals hiv entry inhibitors maraviroc and enfuvirtide scdase and pla2 were purchased from sigmaaldrich hiv fusion peptide was customsynthesized by the yale wm keck biotechnology resource laboratory

hiv pseudovirus production hiv env particles pseudotyped with envelope gp160 protein and containing mlv core proteins were prepared by cotransfection of 293t cells with 3 mg of pfbluc mlv vector plasmid 2 mg of phit60 packaging construct 1 mg of mlv mkogag and 3 mg of hiv jrfl env plasmids using the polyfect reagent qiagen 16 the viruscontaining medium was collected at 48 hours after transfection and centrifuged at 2500 rpm for 10 min at 4c to clear debris to prepare didlabeled hiv particles 293t medium was replaced with optimem i gibco life technologies containing 10 mm did at 16 to 20 hours after transfection after 4 hours of incubation at 37c unincorporated dye was removed by washing and cells were returned to regular growth medium after 24 hours of incubation the extracellular medium was collected centrifuged at low speed to remove cell debris filtered through a 045mm syringe filter and stored at 80c until use for control experiments mlvs pseudotyped with vsvg and mlv core proteins were also prepared for bulk binding and lipid mixing assays membranes of viral particles were labeled with 20 ml of octadecyl rhodamine b chloride 1 mgml r18 in 50 ml of virus for 60 min at room temperature and then free r18 was removed on a sephadex pd10 desalting column ge healthcare the infectious titer was determined by a luciferase assay as previously described 42 in cd4 ccr5 hela cells for hiv env pseudotyped viruses

preparation of gpmvs derived from cd4 ccr5 hela cells cd4 ccr5 hela cells were provided by d m rekosh university of virginia gpmvs were produced and isolated from the cells as previously described 17 18 with slight modifications briefly cd4 ccr5 hela cells were cultured in iscoves modified dulbeccos medium with 10 fetal bovine serum fbs g418 02 mgml 1 penicillinstreptomycin penstrep and puromycin 1 mgml in an incubator at 37c with 5 co 2 plain cd4 ccr5 hela cells which lack cd4 and ccr5 were grown in dulbeccos modified eagles medium 10 fbs and 1 penstrep in the same incubator after growing the cells to a confluence of 70 to 90 in culture dishes cells were washed three times with blebbing buffer [10 mm hepes 150 mm nacl and 2 mm cacl 2 ph 74] membrane vesiculation was then induced by adding 25 mm formaldehyde and 2 mm dtt or 2 mm nem in ordered membrane domains in resting t cell plasma membranes are nanoscopic and shortlived but small dynamic domains can coalesce to create larger ones to function as signaling platforms upon pathogen invasion helper cd4 t cells recognize the pathogenderived antigens on the surface of antigenpresenting cells apc and become activated by coalescence of membrane domains the activation of cd4 t cells stimulates the ability of b cells and cd8 t cells to defend against the invading pathogens in this model we propose that cd4 t cells that are challenged by pathogens are more prone to hiv infection than resting t cells as indicated by the red arrows the hiv infection leads to apoptotic t cell death and ultimately results in the progression to aids

blebbing buffer for 1 hour at 37c following shaking solutions containing celldetached gpmvs were gently decanted into a 50ml tube the gpmvs were placed on ice for 30 min to settle relatively large gpmvs 10 mm in diameter which were then transferred from the bottom of the tube to coverslips for observation on an epifluorescence microscope

lateral distribution and relative amount of cd4 and ccr5 on gpmvs we observed the lateral distributions of cd4 and ccr5 on cellattached and isolated gpmvs for the preparation of cellattached gpmvs cd4 ccr5 hela cells were seeded on quartz slides or 18mm glass coverslips and grown to about 40 confluence after inducing gpmvs from the cells as described above they were rinsed extensively with phosphatebuffered saline pbs to remove gpmvinducing chemicals cellattached gpmvs or isolated gpmvs were blocked with 01 bovine serum albumin in pbs and then the vesicles were stained with the ld marker dii 02 mm or dio 05 mm andor the lo marker alexa 555conjugated ctxb 1 mgml for 60 min on ice after labeling the gpmvs were incubated with an anticd4 antibody andor an anticcr5 antibody conjugated with alexa 488 andor alexa 647 respectively at a concentration of 5 mgml at 4c for 60 min the lateral distribution of fluorescent lipids and proteins on cells or gpmvs was visualized using an epifluorescence microscope the immunofluorescence staining was also used to evaluate the amount of cd4 and ccr5 on the surface of gpmvs for quantification isolated gpmvs were incubated with antibodies at 4c for 60 min and then unbound antibodies were removed by centrifugation 16000g for 30 min at 4c gpmv pellets were resuspended in 400 ml of hepes buffer relative amounts of cd4 and ccr5 were measured at room temperature by fluorescence emission intensities of alexa 488 at 520 nm and alexa 647 at 667 nm in a jobinyvon fluorolog3 spectrofluorometer jobinyvon with excitation wavelength at 495 and 650 nm respectively gpmvs from cd4 ccr5 hela cells were used as negative controls and displayed only low or negligible fluorescence intensity

single and bulk binding assay of hiv env particles to gpmvs for the binding of single hiv env particles to gpmvs gpmvs were induced from cd4 ccr5 hela cells on quartz or glass slides after staining with dio or 7nitro213benzoxadiazol4yl nbddope gpmvs were rinsed with buffer in a fabricated flowthrough chamber hiv particles labeled with mko mkogag were injected into the chamber containing the cellattached or isolated gpmvs and incubated at 4c for 60 min binding of the particles to gpmvs was visualized by epifluorescence microscopy for binding sites of virions and the number of virions bound to gpmvs was counted for binding efficiency binding sites of vsvg env particles on gpmvs were also performed for control experiments to quantify bulk binding efficiency of hiv r18labeled viral particles were added to gpmvs labeled in a tube with nbddope and incubated at 4c for 60 min unbound particles were removed by centrifugation 16000g for 30 min at 4c the pellets containing virusbound gpmvs were resuspended in 400 ml of 05 vv triton x100 fluorescence emission intensities of r18 at 590 nm and nbd at 535 nm were recorded at room temperature in a jobinyvon fluorolog3 spectrofluorometer jobinyvon with excitation wavelength at 560 and 460 nm respectively the ratio of fluorescence intensity at 590 nm535 nm was used to measure relative binding efficiencies of hiv env particles to gpmvs

fusion between r18labeled virus particles and unlabeled gpmvs was measured by the dequenching of r18 fluorescence unlabeled gpmvs were added to the r18labeled virions 1 10 8 particles in hepes buffer at room temperature concentration of gpmvs was estimated by total protein concentration in gpmvs using a bicinchoninic acid assay protein concentrations of gpmvs derived from cd4 ccr5 mbcdtreated cd4 ccr5 and plain cd4 ccr5 hela cells were 101 87 and 62 mgml respectively fluorescence intensities were measured under constant stirring using a fluorolog3 spectrofluorometer jobinyvon with excitation and emission wavelengths of 555 and 590 nm respectively dequenching of r18 fluorescence was normalized to the initial fluorescence ff 0

to prepare the samples for electron cryomicroscopy cryoem hiv env particles were incubated with gpmvs for 60 s at room temperature to capture early stages of membrane fusion between hiv particles and gpmvs samples 35 ml were applied to either cflat or quantifoil holey carbon grids manually blotted to near dryness with filter paper and rapidly plunged into a slurry of liquid ethane the grids were then transferred under liquid nitrogen to a tecnai f20 cyroem philipsfei operating at 120 kv images were recorded at magnifications of 11000 or 30000 under lowelectron dose conditions 20 e 2 using a 4k 4k chargecoupled device ccd camera gatan

for cholesterol depletion isolated gpmvs from cd4 ccr5 hela cells were incubated with 5 mm mbcd in hepes buffer for 30 min at room temperature other gpmvs were incubated with 5 mm lysosm or 20 mm lysopc in hepes buffer for 30 min at room temperature to examine the effect of lysolipids on lipid phases yet other gpmvs were added to 10 u of pla2 in 10 mm trishcl buffer ph 85 or 10 mu of scdase in 50 mm sodium acetate buffer ph 60 which was adjusted to an osmolality of 300 mmolkg by the addition of nacl and incubated at room temperature for 30 min after incubation gpmvs were isolated by centrifugation 16000g for 30 min at 4c and were used for measurements of hiv binding and fusion

fluorescence images were recorded on a zeiss axiovert 200 fluorescence microscope carl zeiss wi"

h a mercury lamp as a light source 40 or 63 water immersion objectives carl zeiss numerical aperture 095 and an electronmultiplying ccd cooled to 70c ixon dv887escbv andor as a detector images were acquired using homemade software written in labview national instruments membranes stained with dio or nbd were epiilluminated through a 480nm bandpass filter d48030 chroma and via a dichroic mirror 505dclp chroma fluorescence was observed through a 535nm bandpass filter d53540 chroma diior rhodaminestained membranes and mkolabeled viral particles were epiilluminated through a 540nm bandpass filter d54025 chroma and via a dichroic mirror 565dclp chroma fluorescence was observed through a 605nm bandpass filter d60555 chroma viral particles or luvs stained with did were illuminated through a 620nm bandpass filter et62060 chroma and observed through a 665nm bandpass filter hq66560 chroma all fluorescence imaging was performed at room temperature 22c images were processed and assessed for colocalization using imagej

guvs were prepared via the electroformation technique briefly 25 ml of a 10 mm lipid mixture composed of bsmbpcbpscholesterol 2111 containing the fluorescent lipid probe rhpe [01 mole percent mol ] was deposited on the conducting surfaces of two indium tin oxidecoated glass slides after the slides were placed in a vacuum desiccator for 90 min to remove residual solvent a fabricated chamber composed of two conducting slides facing each other separated by a 05mm spacer was filled with 300 mm sucrose in h 2 o guvs were generated by applying alternating electric current 3 v 10 hz through a function generator for 120 min at 60c and transferred into a 300 mm glucose solution to settle by gravity on the microscope slide

lipid mixing of luvs mediated by hiv fusion peptide luvs were prepared by extrusion phospholipids composed of bsm bpcbpscholesterol 2111 were dissolved in a mixture of chloroform and methanol and the solvent was evaporated under a stream of nitrogen gas in a glass test tube the thin lipid film was further dried overnight under vacuum and hydrated with 05 ml of hepes buffer [10 mm hepes and 120 mm nacl ph 72] the suspension was vigorously vortexed for 5 min was subjected to 10 freezethaw cycles and was then extruded 21 times through two stacked polycarbonate filters with a pore size of 100 nm avestin the lipid mixing assay was based on fluorescence resonance energy transfer between nbdpe and rhodaminepe the hiv fusion peptide was added to 50 mm luvs with a ratio of 19 of labeled 1 mol of nbdpe and rhodaminepe each to unlabeled luvs in hepes buffer at room temperature the fluorescence was recorded under constant stirring in a fluorolog3 spectrofluorometer jobinyvon with the excitation and emission wavelengths set at 460 and 535 nm respectively the value for 0 lipid mixing was the fluorescence intensity of the luv suspension before the fusion peptide was added whereas the value for 100 lipid mixing was obtained by adding triton x100 [final concentration 05 vv] to the suspension at the end of each experiment

quartz slides were cleaned by boiling in contrad detergent for 15 min and by sonication in a hot bath for 30 min after rinsing with deionized water and ethanol the slides were immersed in piranha solution 31 mixture of 95 sulfuric acid and 30 hydrogen peroxide to remove remaining organic residues followed by extensive rinsing in pure water the first leaflet of the sppm was prepared by langmuirblodgett lb transfer directly onto the quartz slide lipid mixtures composed of bsm bpccholesterol 221 with 3 mol of dmpe 12dimyristoylsnglycero3phosphoethanolaminepolyethyleneglycoltriethoxysilane 43 were spread onto a pure water surface in a nima 611 lb trough ksv nima after allowing the solvent to evaporate for 10 min the monolayer was compressed at a rate of 10 cm 2 min to reach a surface pressure of 32 mnm immediately before use quartz slides were further cleaned for 10 min in an argon plasma sterilizer harrick scientific and then dipped into the trough at a speed of 200 mmmin and withdrawn at 5 mmmin while keeping the monolayer surface pressure constant at 32 mnm the transferred lipid monolayer was dried in a vacuum desiccator at room temperature overnight and cured in a 70c oven for 40 min to covalently link the polymer to the sio 2 slide surface after equilibration in the desiccator to room temperature the slide with the tethered polymersupported lb monolayer was placed in a custombuilt flowthrough chamber a suspension of gpmvs in hepes buffer was injected into the chamber and incubated for at least 2 hours at room temperature to form the distal leaflet of the sppm bilayer excess unfused gpmvs were then washed out by extensive rinsing with hepes buffer

the integrity of the sppms and the diffusion of the lipids within them were examined by fluorescence recovery after photobleaching frap bilayers were bleached in a pattern of parallel stripes and the data were fit to the model ft f f 0 f expda 2 t where f 0 and f are the initial and final fluorescence intensities after bleaching respectively a 2pp p is the stripe period 127 or 32 mm and d is the lateral diffusion coefficient the mobile fraction mf which reflects the percentage of observed fluorescence recovery within the time frame of a frap experiment 1 min is given by mf ff0 f pre f 0 200 where f pre is the fluorescence intensity before photobleaching ten regions on three independently prepared bilayers were sampled to determine the reported average values

binding and fusion of single hiv env particles on sppms by tirf microscopy to measure binding to sppms mkolabeled mkogag hiv env particles were injected into a chamber with unlabeled sppms and the fluorescence increase by hiv binding to sppms was monitored over time by prismbased tirf microscopy the prismquartz interface was lubricated with glycerol to allow easy translocation of the sample cell on the microscope stage the light source was an argon ion laser innova 90c coherent emitting light at 514 nm and fluorescence was observed through a 610nm bandpass filter d61060 chroma the beam was totally internally reflected at an angle of 72from the normal to produce an evanescent wave the intensity of the laser beam was computercontrolled through an acoustooptic modulator isomet or could be blocked entirely by a computercontrolled shutter the laser intensity shutter and camera were controlled by a homemade program written in labview national instruments to investigate the targeting of hiv particles to different regions of ldlo phaseseparated sppms mkolabeled particles were incubated with sppms that were stained with dio for 60 min at room temperature after unbound particles were washed away with hepes buffer phaseseparated sppms were visualized by epifluorescence microscopy and bound particles were visualized by tirf microscopy to analyze and quantify the distribution of sppmbound particles we distinguished three regions of the sppm lo domains ld phase areas and lold boundary regions with a 075mm width centered on the perimeter of each lo domain custombuilt particletracking software 35 was used to automatically detect the position x and y coordinates of each particle to monitor the fusion of single hiv particles with the sppm didlabeled hiv env particles were injected into a chamber one large window wall of which was formed by the sppm the light source for tirf illumination was a diode laser cube640 coherent to excite the lipid dye did through a 620nm filter et62060 chroma and via a dichroic mirror 660dclp chroma did fluorescence was observed through a 665nm bandpass filter hq66560 chroma data acquisition and image analysis were accomplished through custombuilt software written in labview national instruments 35 single events that included docking hemifusion and full fusion were measured by analyzing the peak fluorescence intensities from each bound particle as a function of time events with no fluorescence change over time were classified as docking those with decays to around onehalf of the original peak intensity were classified as hemifusion and those with complete decays were classified as direct full fusion events 44

supplementary material for this article is available at httpadvancessciencemagorgcgi contentfull36e1700338dc1 fig s1 association of cd4 and ccr5 with lipid rafts in cd4 ccr5 hela cells with stably expressed cd4 and ccr5 fig s2 formation of gpmvs by treatment of cd4 ccr5 hela cells with small amounts of formaldehyde and dtt fig s3 partitioning of cd4 and ccr5 in gpmvs induced by nem instead of formaldehyde and dtt from cd4 ccr5 cells fig s4 hiv env pseudovirus particles bind preferentially at boundaries between coexisting lo and ld domains in gpmvs fig s5 vsvg pseudovirus particles bind to ld membrane regions in gpmvs fig s6 electron cryomicrographs of hiv env particles boundfused to gpmvs fig s7 modulation of lipid phases does not affect binding of hiv to gpmvs fig s8 preparation of sppms with gpmvs fig s9 lateral distribution of cd4ccr5 in sppms movie s1 growing gpmvs on cell surfaces " "Yang

Sung-Tae. Kreutzberger

Alex J. B...." HIV virions sense plasma membrane<br>heterogeneity for cell entry Sci Adv " It has been proposed that cholesterol in host<br>cell membranes plays a pivotal role for cell entry of<br>HIV. However

it remains largely unknown why<br>virions prefer cholesterol-rich heterogeneous<br>membranes to uniformly fluid membranes for membrane<br>fusion. Using giant plasma membrane vesicles<br>containing cholesterol-rich ordered and<br>cholesterol-poor fluid lipid domains

we demonstrate that the<br>HIV receptor CD4 is substantially sequestered<br>into ordered domains

whereas the co-receptor CCR5<br>localizes preferentially at ordered/disordered domain<br>boundaries. We also show that HIV does not fuse from within<br>ordered regions of the plasma membrane but rather at<br>their boundaries. Ordered/disordered lipid domain<br>coexistence is not..." 154 6872

92142e072745fbab996c5861e5974aa03805398a macrophages found in circulating blood as well as integrated into several tissues and organs throughout the body represent an important first line of defense against disease and a necessary component of healthy tissue homeostasis additionally macrophages that arise from the differentiation of monocytes recruited from the blood to inflamed tissues play a central role in regulating local inflammation studies of macrophage activation in the last decade or so have revealed that these cells adopt a staggering range of phenotypes that are finely tuned responses to a variety of different stimuli and that the resulting subsets of activated macrophages play critical roles in both progression and resolution of disease this review summarizes the current understanding of the contributions of differentially polarized macrophages to various infectious and inflammatory diseases and the ongoing effort to develop novel therapies that target this key aspect of macrophage biology "1 macrophages m represent a ubiquitous yet complex and nuanced population of immune cells that play essential roles in both disease and homeostasis throughout the body hume 2008 in addition to monocytes and m circulating throughout the bloodstream specialized tissueresident m can be found in most major organs including kupffer cells in the liver langerhans cells in the skin microglia in the brain splenic red pulp m lung alveolar m adipose tissue m and bone osteoclasts to name a few davies et al 2013 gautier et al 2012 ji et al 2013 you et al 2013 while some identify these populations as the endpoint of bone marrow monocyte maturation several lines of evidence indicate that tissue resident m originate during embryogenesis in association with their specific tissue independently from blood monocytes and monocytesm recruited to sites of inflammation davies et al 2013 gomez and geissmann 2013 schulz et al 2012 regardless of their location m are responsible for the maintenance of healthy tissues through phagocytic clearance of apoptotic cells and foreign materials and through tissue repair and remodeling during wound healing duffield 2005 ghavami et al 2014 majai et al 2014 mantovani et al 2013 m are also major regulators of the inflammatory response to disease and infection acting as a bridge between innate and adaptive immunity by monitoring the microenvironment through an array of surface receptors and secreting appropriate cytokines and chemokines heydtmann 2009

depending on the stimuli they encounter tissue resident and circulatory m populations can be directed to distinct phenotypic programs in a process known as m polarization fig 1 table 1 the diverse properties of different m subsets can have drastic effects on health and disease within the tissues where they reside while the induction of a particular subset can be protective during homeostasis or disease this process can be altered or subverted to enhance pathogenesis and disease progression by for example inappropriately dampening the immune response or exacerbating harmful inflammation murray and wynn 2011 therefore this review aims to summarize recent findings regarding the identity properties and roles of polarized m in various disease models and the development of therapeutic strategies that target both the process of m polarization and individual m subsets

the most welldescribed and commonly reported paradigm of m polarization is the m1m2 polarization axis mantovani et al 2004 martinez et al 2009 sica et al 2013 originally named to reflect relationships to th1th2 polarization of immune responses m1 and m2 m are also referred to as classically or alternatively activated m respectively gordon 2003 mills et al 2000

classical activation is stimulated by microbial products and proinflammatory cytokines ifn andor lps or tnf and the resulting m1 m are characterized by high antigen presentation high production of il12 and il23 and high production of nitric oxide no and reactive oxygen intermediates roi verreck et al 2004 m1 m have been shown to produce several other inflammatory cytokines like tnf il1 6 and 12 type i ifn cxcl13 5 and 810 ccl25 and 11 cxcl16 and cx3cl1 mantovani et al 2004 sica and mantovani 2012

by contrast alternativem2 activation is mediated by il4 il table 1 sr scavenger receptor mr mannose receptor ho1 heme oxygenase1 vegf vascular endothelial growth factor sd1 sulfiredocin1 trreductase thioredoxinreductase kadl et al 2010 leitinger and schulman 2013 murray and wynn 2011 polarization state gene expression cytokines chemokines m1 cd80 cd86 mhc iii il1r i tlr2 tlr4 inos tnf il1 il6 il12 il15 il23 ros inos type i ifn cxcl13 cxcl5 cxcl810 cxcl16 ccl25 ccl8 ccl11 ccl15 ccl20 cx3cl1 m2a cd163 mhc ii sr cd206 mr il1r ii ym1 fizz1 arg1 il10 tgf il12 il1ra ccl1 ccl2 ccl24 10 and il13 which were initially thought to produce deactivated m martinez et al 2009 m2 m are marked by the upregulation of several surface molecules including dectin1 dcsign mannose receptor mrc1cd206 scavenger receptor a cd204 scavenger receptor b1 cd163 ccr2 cxcr1 and cxcr2 gordon 2003 mantovani et al 2004 martinez et al 2009 m2 m exhibit altered cytokine and chemokine production and typically produce high levels of il10 and low levels of il12 mosser 2003 ccl1 ccl2 ccl17 ccl18 ccl22 ccl24 and il1ra are also made by alternatively activated m mantovani et al 2004 genetic studies of m2 m in mouse models have identified additional signatures of alternative activation including arginase 1 arg1 ym1 a member of the chitinase family and fizz1 found in inflammatory zone 1 retnla raes et al 2002 2005 generally the m2 polarization state is characterized by little to no secretion of proinflammatory cytokines increased secretion of antiinflammatory cytokines enhanced scavenging of cellular debris promotion of tissue remodeling and repair and in some cases increased capacity to fight parasitic infections additionally the concept of resolution of inflammation has evolved and is no longer perceived as a passive process that simply occurs when the insult disappears but rather as a highly orchestrated response coordinated by a complex regulatory network of cells and antim1 mediators called proresolving mediators rius et al 2012 m2 m can be further divided into subtypes according to their inductive stimuli and secreted chemokines martinez et al 2008 m2a m are stimulated by il4 and il13 and produce ccl24 ccl22 ccl17 and ccl18 which are recognized by ccr3 ccr4 and ccr8 and promote recruitment of eosinophils basophils and th2 cells m2b m result from activation with immune complexes and tlr agonists like lps and produce ccl1 which recruits tregs il10 drives m polarization to m2c cells which produce ccl16 and 18 recruiting eosinophils and nave t cells respectively m2d m accumulate in the tumor microenvironment and present an il10 hi vegf hi m2 profile but also exhibit some m1 characteristics such as expression of infinducible chemokines ccl5 cxcl10 and cxcl16 duluc et al 2009 a full understanding of the m1m2 paradigm of m polarization however contains some caveats first m1 and m2 m as defined in the foundational literature most likely do not exist as distinct categories but rather should be considered as extremes at either end of a continuum of overlapping functional states mosser and edwards 2008 indeed m with combinations of m1 and m2 markers can be found in atherosclerotic plaques and some murine tumors kadl et al 2010 umemura et al 2008 second unlike the irreversible phenotypic changes seen in lymphocytes after exposure to polarizing cytokines m polarization is both transient and plastic biswas and mantovani 2010 biswas et al 2008 sica et al 2013 stout and suttles 2004 for example m2 m can be reprogrammed to express m1 genes following exposure to tlr ligands or ifn mylonas et al 2009 stout et al 2005 finally while there is partial overlap of m1and m2identifying markers in murine and human studies there are still markers in each system that fail to translate to the other the chitinaselike proteins ym1 and ym2 along with fizz1 are markers of murine m2 polarization which lack human orthologs while ccl14 ccl18 and ccl23 are humanrestricted m2 markers with no murine orthologs chang et al 2001 martinez et al 2009 raes et al 2002 finally there are other specially activated m m4 mhem and mox that have been described in atherosclerosis and may lie on a separate activation axis from m1m2 m fig 1 these atherosclerotic m subsets have been discussed in recent reviews fenyo and gafencu 2013 leitinger and schulman 2013 but will not be examined in detail here

the network of molecular mediators that regulate m1m2 polarization in response to various stimuli is incompletely understood but several signaling pathways have been implicated in this process fig 2 one of the major pathways identified is the jakstat pathway which mediates responses to a collection of different cyotkines and growth factors and regulates processses from hematopoiesis and immune development to lactation and adipogenesis rawlings et al 2004 binding of ifn to its cell surface receptor leads to activation of receptorassociated jaks which in turn cause stat1 to dimerize and translocate to the nucleus where it initiates transcription of genes that promote m1associated functions like enhanced microbicidal activity and proinflammatory cytokine production hu et al 2007 rauch et al 2013 mspecific deletion of socs3 an inhibitor httpmolcellsorg

mol cells 277 of cytokine and jakstat signaling was found to increase levels of the m1 genes il1 il6 il12 il23 and inos qin et al 2012a and increase phosphorylation of stat1 and stat3 qin et al 2012b in contrast stat6 is activated by il4 and il13 to induce m2 polarization daley et al 2009 moreno et al 2003 stolfi et al 2011 cjun nterminal kinase jnk a mitogenactivated protein kinase mapk involved in cell proliferation transformation differentiation and apoptosis is likely involved in this pathway upon activation jnk can phosphorylate serine 707 on stat6 thereby deactivating it shirakawa et al 2011 a study of m polarization in obesity showed that mice lacking the jnk activator mlk3 were also deficient in m1 m polarization gadang et al 2013 the transcription factors ppar and ppar are activated by stat6 and necessary for m2 polarization and ppar m exhibit enhanced activation of jnk following treatment with adipocyteconditioned medium which contains the m2 cytokines il4 and il13 kang et al 2008 odegaard et al 2007 the zincfinger transcriptional regulator krppellike factor 4 klf4 is involved in this pathway as well and cooperates with stat6 to skew polarization towards m2 by sequestering coactivators of nfb liao et al 2011

furthermore the phosphoinositol3kinase pi3k signaling pathway which activates multiple kinase cascades through the production of the second messenger pip3 regulates m survival and gene expression via activation of the akt family of serinethreonine protein kinases luyendyk et al 2008 knockout studies have demonstrated that m1 polarization depends on the activation of akt2 while m2 polarization requires akt1 arranz et al 2012 in addition the pi3kakt signaling pathway controls the activation of mtor which promotes m2 polarization byles et al 2013 mercalli et al 2013 weichhart and semann 2008

interferonregulatory factor irf proteins are also regulators of m polarization irf5 is associated with m1 polarization and promotes the transcription of genes encoding il12 while repressing the gene that encodes il10 krausgruber et al 2011 notch signaling through the nuclear transducer rbpj controls expression of irf8 which induces m1 gene expression xu et al 2012 irf4 is highly expressed in adipose tissue m atm and its deletion leads to increased production of il1 and tnf and expression of m1 markers indicating that irf4 activation contributes to m2 polarization eguchi et al 2013 the irfs also underlie the ability of gmcsf and mcsf to induce polarization gmcsf leads to downstream activation of irf5 m1 while mcsf leads to irf4 m2 activation lawrence and natoli 2011

given the ability of m to acquire enhanced microbicidal abilities following stimulation with microbial products and the preeminent roles of m in both innate and adaptive immune responses one might predict that pathogens would evolve strategies to redirect and alter m activation in their favor several transcriptome analysis studies have established that innate immune cells particularly m engage in a common response to pathogen challenge that involves a shared pattern of gene expression jenner and young 2005 nau et al 2002 a multistudy review of transcriptional responses of mononuclear phagocytes to bacteria and bacterial components focusing specifically on genes involved in m polarization identified a common response program that mainly involved the upregulation of m1associated genes including the cytokines tnf il6 il12 il1 the cytokine receptors il7r and il15ra the chemokines ccl2 ccl5 and cxcl8 and the chemokine receptor ccr7 benoit et al 2008 this m1 activation program is typically associated with protection against disease and m1 polarization has been shown to aid host control of several bacteria including listeria monocytogenes salmonella typhimurium mycobacterium tuberculosis mycobacterium ulcerans and chlamydia infections benoit et al 2008 chacnsalinas et al 2005 jouanguy et al 1999 kiszewski et al 2006 rottenberg et al consequently several pathogenic bacteria especially intracellular species have developed mechanisms to interfere with m polarization in order to enhance their own survival some species accomplish this by blunting m1 polarization to reduce inflammation and microbicidal functions of m the intracellular form of the enteropathogen shigella flexneri produces an altered hypoacetylated form of lps that evades recognition by tlr4 and elicits decreased production of proinflammatory cytokines from murine bone marrow derived m bmdm paciello et al 2013 during pulmonary infection in mice staphylococcus aureus induces akt1 signaling to enhance socs1 activity and inhibit nfb activity shifting m from an antimicrobial m1 phenotype to a functionally inert one m tuberculosis secretes the virulence factors lipoarabinomannan and early secretory antigenic target6 esat6 which inhibit m1 activation by inhibiting phagosome maturation and nfb activation respectively lugovillarino et al 2011 m tuberculosis also subverts the inflammatory response by stimulating wnt6 signaling in infected m in granulomatous lesions in the lung driving m2like polarization schaale et al 2013 s aureus biofilms are resistant to m invasion but those m that do successfully penetrate catheterassociated biofilms in vitro display decreased expression of il1 tnf and inos expression but robust arg1 expression signifying an m2 profile thurlow et al 2011 s typhimurium has been shown to preferentially associate with m2 m and ppar expression is upregulated in salmonellainfected m while ppar deficiency severely inhibits bacterial replication and persistence eisele et al 2013 interestingly the dependency of s typhimurium on ppar expression was shown to be due to its metabolic effects rather than its ability to reduce production of antimicrobial mediators by promoting m2 polarization and it remains to be determined whether s typhimurium directly augments ppar activity to promote persistence

similar to evasion strategies employed by bacterial pathogens many viruses take advantage of the m polarization system to enhance their own growth and virulence however unlike bacterial pathogens which generally tend to thrive within and encourage production of m2polarized m viral pathogens more commonly activate m1 polarization this inflammatory phenotype is often correlated with disease severity hepatitis c virus preferentially infects hepatocytes and establishes a chronic inflammatory infection often leading to fibrotic cirrhosis and hepatocellular carcinoma hcc lavanchy 2011 it has been demonstrated that the viral protein ns3 enhances il12 and tnf production by thp1 m implicating m1 polarization in sustaining inflammation hajizadeh et al 2013 furthermore activation of m with tlr agonists triggers the secretion of tnf which promotes hcv entry into polarized hepatoma cells by relocalizing the tight junction protein and hcv entry factor occludin fletcher et al 2013 of the three common clades of avian h5n1 influenza virus circulating in poultry in china 232 234 and 7 clade 234 is the most successful at infecting replicating within and inducing cytopathic effects in human monocytederived m mdm h5n1 clade 234 also stimulated the highest expression of il1 il6 il8 tnf ifn and mcp1 in mdms m2 m polarization by s aureus which is commonly present among the airway mucosal microbiota inhibits influenzamediated lung injury implying that m1 m exacerbate flu infection

nonetheless some viruses do benefit by skewing m polarization towards an m2 phenotype during infection by severe acute respiratory syndrome coronavirus sarscov lung damage resulting from both intrinsic viral infection and dysregulation of the host immune response rapidly progresses to diffuse alveolar damage resulting in acute respiratory distress syndrome and pulmonary fibrosis franks et al 2003 peiris et al 2003 a recent study revealed that sarscovinfected mice lacking hematopoeitic stat1 expression have greater weight loss and lung pathology associated with upregulation of the m2 indicators ym1 fizz1 il4 and il13 page et al 2012 absence of lung disease and prefibrotic lesions in infected stat1stat6 doubleknockout mice also supported the notion that m2 m contribute to sarscov pathogenesis human cytomegalovirus hcmv has a more complex relationship with m polarization the hcmv gene ul111a encodes a homolog of human il10 that is capable of polarizing monocytes towards an antiinflammatory m2c phenotype including high expression of the scavenger receptor cd163 suppression of mhc expression and exppression of heme oxygenase 1 which suppresses tnf and il1 avdic et al 2013 additionally hcmv optimally infects m2but not m1polarized m and latephase hcmv infection is dependent on the m2promoting activation of mtor poglitsch et al 2012 despite this hcmvactivated m have been shown to adopt an m1 transcriptome profile chan et al 2008 hiv1 similarly seems to benefit from m2 polarization hiv1 displays impaired viral dna synthesis delayed proviral integration and reduced proviral transcription in m1 m while the m2a surface receptor dcsign facilitates hiv1 entry dna synthesis and transmission from infected m to cd4 t cells cassol et al 2010 notably clathrinmediated endocytosis of hiv1 is increased in m1 and decreased in m2 m but this method of endocytosis leads to increased viral degradation and is unlikely to result in productive infection gobeil et al 2012 yet like hcmv hiv1 infection of mdms drives them toward m1 polarization and the viral protein nef is preferentially taken up by m2 m and stimulates an m2tom1 transition cassol et al 2010 chihara et al 2012 lugovillarino et al 2011 these contradictions may be explained by a viral survival strategy that takes advantage of both m1 and m2 m as means to different ends m2 m as a reservoir of replication and m1 m to recruit fresh immune cells to spread the infection this can also be inferred from the ability of proinflammatory cytokines and chemokines from hcmvinfected m to enhance virus replication and dissemination smith and bentz 2004a 2004b

type 1 diabetes is an autoimmune disease that results in high blood sugar following the destruction of insulinproducing pancreatic beta cells via activation of innate immunity and expansion of autoreactive t cells and autoantibodyproducing b cells monocytesm from patients with type 1 diabetes present a proinflammatory profile high levels of tnf il6 and il1 when compared to normal subjects bradshaw et al 2009 devaraj et al 2006 shanmugam et al 2004 moreover elevated levels of glucose and islet amyloid polypeptide iapp deposition lead to the activation of tlrs and inflammasomes resulting in beta cell death and decreased insulin secretion henaomejia et al 2013 recently it has been suggested that m1 m may contribute to diabetesrelated complications such as cardiovascular diseases by altering the immune system of type 1 diabetics burke and kolodgie 2004 furthermore the sustained increase of growth hormone in murine models of type 1 diabetes leads to a reduction of diabetes symptoms by attenuating the apoptosis and increasing the expansion of beta cells villares et al 2013 growth hormone also triggers m2 polarization of m via modulation of the cytokine milieu stimulating the activity of suppressor t cells and limiting th17 cell activation villares et al 2013

obesity is a major health problem in western countries and a risk factor for insulin resistance type 2 diabetes hepatic steatosis and artherosclerosis obesity is closely associated with chronic inflammation in adipose tissues suggesting that the chronic excess of nutrients triggers an immune response in adipose tissues goh et al 2011 hotamisligil 2006 kanneganti and dixit 2012 white adipose tissues store energy in the form of fat and regulate systemic metabolism through the release of adipokines by adipocytes that control insulin sensitivity in the liver and skeletal muscle sun et al 2011 tateya et al 2013 in lean subjects and mice adipose tissue m atm present an m2 phenotype and are critical to maintaining insulin sensitivity in adipocytes through il10 production liao et al 2011 lumeng et al 2007a 2007b in metabolic homeostasis m2 atms are maintained by il4 and il13 secreted by adipocytes in a pparand klf4dependent manner wynn et al 2013 zhou et al 2013 in obese subjects and mice adipocytes release proinflammatory mediators ie ccl2mcp1 tnf ccl5 ccl8 and free fatty acid promoting the infiltration of ly6c hi inflammatory monocytes which differentiate into m1polarized atms that express high levels of tnf inos il6 and il1 cinti et al 2005 lumeng et al 2007a olefsky and glass 2010 tateya et al 2013 weisberg et al 2003 weisberg et al 2006 therefore the severity of obesityrelated metabolic dysfunctions correlates with m1 atm infiltration whereas chronic inflammation in adipose tissue inhibits the production of adiponectin thus contributing to the development of insulin resistance in surrounding adipocytes lumeng et al 2007a weisberg et al 2003 2006

recently nafld nonalcoholic fatty liver disease has emerged as an obesityrelated health problem characterized by steatosis accumulation of lipids in hepatocytes hepatic steatosis can evolve to nonalcoholic steatohepatitis nash when accompanied by liver injury ballooning hepatocytes and hepatic inflammation which may be associated with fibrosis and eventually culminates in cirrhosis and hcc the development of the complex pathology of nash involves a variety of liver cells including hepatocytes hepatic m and stellate cells inflammatory mediators especially those derived from adipose tissues the gut and the liver have recently been reported to play a major role in initiating and controlling the progression of nash by regulating lipid metabolism day and james 1998 racanelli and rehermann 2006 tilg and moschen 2010 in particular the activation of innate immune cells such as kupffer cells and infiltrating bloodderived monocytes is a major event of nash development in homeostatic conditions kupffer cells perform immune surveillance by removing pathogens and toxins from the circulation and maintain liver tolerance through il10 secretion thomson and knolle 2010 kupffer cells communicate with a variety of hepatic immune cells and interact directly with hepatocytes passing through the space of disse racanelli and rehermann 2006 in early mouse models of dietinduced steatohepatitis kupffer cells are the first innate cells responding to injured hepatocytes and differentiate toward m1 m promoting the recruitment of bloodderived cd11b int ly6c hi monocytes through secretion of tnf and chemokines mcp1 and ip10 tosellotrampont et al 2012 the recruitment of these inflammatory m1polarized ly6c bloodderived monocytes is dependent of ccr2 and mcp1 karlmark et al 2009 klein et al 2007 miura et al 2012 obstfeld et al 2010 the hallmarks of nash ie steatosis lowgrade inflammation and hepatic recruitment of m1polarized m are reduceddelayed following specific depletion of kupffer cells or by silencing of tnf in myeloid cells tosellotrampont et al 2012 moreover m1polarized kupffer cells also produce inflammatory mediators such as il1 and ros which induce hepatic steatosis and fibrosis miura et al 2012 nfb and jnk activation in kupffer cells may contribute to the development of hepatic inflammation by promoting m1like m polarization

liver m are also implicated in the severity of nash via the expression of tolllike receptors tlr2 tlr4 tlr9 myd88 and scavenger receptors scavenger receptor a and cd36 bieghs et al 2010 miura et al 2010 tlrs and scavenger receptors trigger proinflammatory responses following recognition of hepatic free fatty acids damageassociated molecular pattern damps expressed by steatotic hepatocytes andor bacterial products derived from the gut farrell et al 2012 roh and seki 2013 nash patients show increased intestinal permeability resulting in greater hepatic abundance of bacterial products and other tlr ligands derived from the gut via portal vein circulation the imbalance of gut flora may influence liver disease by activating tlrs expressed on liver cells and leading to the activation of nlpr3 csak et al 2011 farrell et al 2012 henaomejia et al 2013 roh and seki 2013 in models of dietinduced nash and obesity inflammasomedeficient mice develop more severe nash which is fully transferable to wt mice upon prolonged cohousing suggesting that commensal bacteria in the gi tract play an important role in nash disease progression csak et al 2011 henaomejia et al 2012 weisberg et al 2003

m are a highly influential cell type in most varieties of cancer and are recruited to all solid tumors cassetta et al 2011 the contributions of different subsets of polarized m to the tumor microenvironment and cancer progression are therefore a subject of great interest m1 m are generally considered to be beneficial to the host and peritumoral m that express m1 cytokines like ifn il1 and il6 have been shown to have antitumoral effects and are associated with improved prognoses dumont et al 2008 klimp et al 2002 niino et al 2010 berg et al 2002 zhou et al 2010 m1 m may have the opposite effect in virally induced cancers however administration of ifn or tnf to patients infected with kaposi sarcoma virus enhances disease progression monini et al 1999 proinflammatory m are also harmful in intraocular tumors where tnfand inosdependent antitumor responses lead to necrosis of bystander cells and destruction of the eye coursey et al 2012

m2polarized tumorassociated m tam on the other hand have been repeatedly and consistently associated with unfavorable effects like tumor growth angiogenesis and metastasis in malignant cancers the m2 cytokines il4 il13 and il10 are present within the tumor microenvironment and tams from various cancer models have been shown to express an m2 activation profile that includes enhanced expression of cd163 mrc1 ctype lectins il10 and arg1 and decreased production of il12 beck et al 2009 biswas et al 2006 schmieder et al 2012 sica et al 2002 the small secretory lectin reg3 is an important inhibitor of inflammation in pancreatic and intestinal tissues and deficiency of reg3 an activator of the stat3 pathway drastically impairs pancreatic tumor growth by skewing m polarization away from m2 and towards m1 gironella et al 2013 m2 tams have also been shown to increase fibroblastic morphology vimentin and snail expression metalloproteinase activity and proliferation and migration of pancreatic cancer cells implicating them in the development of eptihelialmesenchymal transition and metastasis in hcc high expression of the heparinsulfate proteoglycan glypican3 gpc3 on the surface of cancer cells is associated with increased m infiltration in human patients and humanmouse xenograft transplantation with a gpc3overexpressing cell line leads to infiltration by m expressing m2specific markers takai et al 2009a 2009b m2 tams worsen hcc both by promoting tumor growth and angiogenesis and by encouraging liver fibrosis through il13 and tgf secretion

given that m play important roles in maintaining tissue homeostasis and fighting disease polarized m subsets that specifically contribute to the pathogenesis or amelioration of various diseases present themselves as attractive targets for therapeutic intervention different therapeutic strategies include either targeting the polarized m themselves or manipulating the signaling pathways involved in the process of m polarization to a desirable outcome

bacterial biofilms that form on body surfaces or on surgical implants lead to chronic and recurrent infections and are difficult to treat with antibiotics donlan and costerton 2002 otto 2008 early local administration of m1 m or the c5a receptor agonist ep67 which stimulates m1 polarization significantly attenuated biofilm formation in a mouse model hanke et al 2013 furthermore treatment of established biofilms significantly reduced bacterial burden compared to antibiotic treatment suggesting the potential of a therapeutic alternative hanke et al 2013 microbes themselves may also prove to be useful sources of therapeutics that modulate m polarization in vitro application of extracellular polysaccharide secreted by an oligotrophic bacteria found in lop nur desert bacillus sp lbp32 was found to limit lpsinduced inflammation in the m cell line raw 2647 by inhibiting nfb and jnk activation and may prove useful in diseases characterized by excessive m1 polarization similarly the smallmolecule compound bisnnorgliovictin isolated from the marinederived endophytic fungus s31c inhibits lpsinduced m1 polarization of raw 2647 cells and murine peritoneal m and improves survival in mouse models of sepsis song et al 2013 as a proof of concept for treating inflammatory gastrointestinal diseases a lab strain of e coli was created that secretes a herpes virus homolog of il10 via a secdependent transporter construct viral il10 delivered in this manner was shown to activate stat3 and suppress tnf production in the j7741 murine m cell line frster et al 2013

ikk a downstream mediator of insulin resistance and activator of the nfb pathway and therefore of m1 polarization is inhibited by antiinflammatory salicylates like aspirin which attenuates hyperglycemia and hyperinsulinemia in obese rodents yin et al 1998 yuan et al 2001 several small trials in patients with type 2 diabetes have demonstrated that treatment with salicylates results in a marked reduction of diabetic metabolic parameters and improved glycemic control tateya et al 2013

apolipoprotein ai mimetics are a class of therapeutic molecules that attempt to modulate hdl to treat atherosclerosis and are the subject of extensive clinical and mechanistic study as reviewed in leman et al 2013 interestingly the mimetic d4f also has potential for cancer therapy d4f inhibits the m2associated scavenger receptor cd204sra on tams preventing metastatic spread neyen et al 2013 anticancer therapies also seek to convert protumoral m2 m into m1 m m2 m generated by il6 and prostaglandin e 2 secreted by cervical cancer cells can be repolarized to m1 by coculture with th1 cells and this interaction could possibly be reproduced by activation with cd40l and ifn heusinkveld et al 2011 moreover ifn was shown to successfully switch m2 tams purified from human ovarian tumors to an m1 phenotype and the addition of ifn skewed de novo tumorinduced m2 differentiation of monocytes to favor m1 polarization duluc et al 2009 other potentially therapeutic molecules found to repolarize tams from an m2 to an m1 phenotype include zoledronic acid cpg oligonucleotide and histidinerich glycoprotein coscia et al 2010 huang et al 2012 rolny et al 2011

the integral importance of m in the maintenance of nearly every tissue throughout the body and their position as the first line of defense against many diseases guarantees that they play critical roles in both disease progression and in resolution and that altering the behavior of these cells can mean the difference between healthy recovery and severe illness m polarization itself is an extremely nuanced and finetuned process and can produce nearly infinite variations of endpoint phenotypes each of which has the potential to affect various diseases in different ways while polarized m subsets and the polarization process itself are attractive and novel therapeutic targets in both infectious and inflammatory disease better understanding of how polarization is controlled and how polarized m modulate specific diseases is necessary to fully harness the potential of these strategies

this work was supported by nih grants r01ai098126 and u19ai066328" "Labonte

Adam C.. Tosello-Trampont

Annie-Carole..." The Role of Macrophage Polarization in<br>Infectious and Inflammatory Diseases Mol Cells " Macrophages

found in circulating blood as<br>well as integrated into several tissues and organs<br>throughout the body

represent an important first line of<br>defense against disease and a necessary component of<br>healthy tissue homeostasis. Additionally

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