Abstract Venous malformations (VMs) are localized defects in vascular morphogenesis which can seriously impede or even threaten the patient’s life. VMs are characterized by enlarged, torturous vein-like channels lined by unevenly distributed smooth muscle cells. A large number of mutations in the endothelial TIE2 receptor tyrosine kinase have been found from more than half of the lesions screened, thus providing a common genetic cause. TIE2 has a crucial role in vascular development, remodeling and quiescence. However, the molecular and cellular abnormalities caused by TIE2-mutations in endothelial cells and how they relate to VM formation have been unknown. The aim of this study was to examine how VM-specific mutations affect the molecular characteristics of TIE2-receptor downstream signaling and cellular functions. Because no effective treatment has been available for VMs, a better understanding of the molecular basis of their pathology should enable the development of more potent and non-invasive treatments as well as provide a better understanding of vascular morphogenesis in general. The results demonstrate that the TIE2-VM forms have both common and specific effects on TIE2 and the endothelial cells (ECs) expressing them. Mutation-induced TIE2 autoactivation leading to loss of normal EC monolayer organization due to extracellular matrix (ECM) fibronectin deficiency was found to be a common change. This was shown to occur through chronic activation of the mitogen-activated protein kinase (MAPK) pathway, which also caused activation of the proteolytic plasminogen system. Also, most mutations altered TIE2 trafficking and angiopoietin ligand regulated TIE2 functions, albeit through different mechanisms. Using RNA-screening we showed that the most common sporadic TIE2-VM mutation dysregulates genes affecting vascular development, cell migration and ECM remodeling. PDGFB, a major attractant of vascular mural cells, was found to be strongly attenuated due to chronic activation of Akt, which also increases EC survival, by the TIE2 mutant receptors. To conclude, the results in this thesis reveal genetic, molecular and cellular alterations which may potentiate VM formation. This data provides new information on the pathological mechanisms behind abnormal vascular morphogenesis and should assist the development of new molecular treatment strategies for VM patients. / Tiivistelmä Laskimoepämuodostumat ovat paikallisia verisuoniston kehityksen häiriöitä. Riippuen niiden koosta ja anatomisesta sijainnista ne voivat aiheuttaa merkittävää haittaa. Epämuodostumat koostuvat laajentuneista, laskimonkaltaisista verisuonista, joissa sileiden lihassolujen kerros on puutteellisesti järjestäytynyt. Yli puolessa tutkituista laskimoepämuodostumista havaitaan mutaatioita verisuonten sisäpinnan endoteelisoluissa ilmenevässä TIE2 reseptorityrosiinikinaasissa, joka säätelee verisuonten kehitystä, muokkausta ja fysiologista toimintaa. TIE2-mutaatioiden aiheuttamia molekyyli- ja solutason muutoksia tai niiden yhteyttä epämuodostumien syntyyn ei ole aikaisemmin tunnettu. Tämän tutkimuksen tarkoituksena oli selvittää, miten laskimoepämuodostumista löydetyt mutaatiot vaikuttavat TIE2-reseptorin toimintaan molekyyli- ja solutasolla sekä TIE2-reseptorista alkavaan solunsisäiseen viestintään. Koska pysyvää hoitomuotoa laskimoepämuodostumille ei tunneta, voisi tieto niiden taustalla olevista patologisista mekanismeista edesauttaa parempien, ei-kajoavien hoitomuotojen kehittämisessä ja antaa myös yleisesti uutta tietoa verisuoniston kehityksestä. Väitöskirjan tulokset osoittavat, että mutaatiot vaikuttavat TIE2-reseptoriin ja sitä ilmentäviin endoteelisoluihin mutaatioille yhteisillä sekä mutaatiokohtaisilla tavoilla. Mutaatioille tyypillinen TIE2-reseptorin ligandista riippumaton aktivaatio aiheutti aktivaation nousun myös TIE2:sta alavirtaan olevissa viestinvälittäjissä. Tämä puolestaan johti fibronektiini-proteiinin häviämiseen soluväliaineesta, sileitä lihassoluja säätelevän PDGFB-kasvutekijän ilmenemisen laskuun ja solujen ohjelmoidun solukuoleman vähenemiseen. Useimmat tutkitut mutaatiot muuttivat myös TIE2-reseptorin sijaintia soluissa häiriten TIE2:n angiopoietiini-ligandien säätelemiä toimintoja usean eri mekanismin kautta. Transkriptomin laajuiset RNA-tutkimukset osoittivat monien verisuonten kehitykseen, solujen liikkumiseen ja soluväliaineen muokkaukseen liittyvien geenien ilmentymisen muuttuneen. Lopputuloksena tutkimus paljasti geeni-, molekyyli-, ja solutason muutoksia, jotka saattavat vaikuttaa laskimoepämuodostumien syntyyn. Tulokset antavat lisätietoa sairautta aiheuttavista mekanismeista verisuoniston kehityksen häiriöiden taustalla ja ovat hyödyksi kehitettäessä uusia lääkkeitä laskimoepämuodostumien molekulaarisia hoitoja varten.

Les cellules béta pancréatiques synthétisent et sécrètent l’insuline, unique hormone hypoglycémiante de l’organisme. Ces cellules jouent un rôle central dans l’apparition du diabète, préserver leurs masses fonctionnelles est donc essentiel. Le récepteur GLP-1, appartenant à la classe B de la super famille des récepteurs couplés aux protéines G (RCPGs), est considéré comme une cible thérapeutique majeure dans le traitement du diabète de type 2. Via son récepteur, le GLP-1 potentialise la sécrétion d’insuline en réponse au glucose et favorise la survie des cellules beta. Les glucocorticoïdes sont des hormones du stress impliquées dans la régulation énergétique, largement utilisés en thérapeutique pour leur propriétés anti-inflammatoire, immunosuppresseur et antiallergique. Néanmoins, les glucocorticoïdes administrés en chronique sont diabétogènes en exerçant notamment des effets délétères sur les cellules beta. Nous avons caractérisé l’impact d’une exposition prolongée des cellules beta à un glucocorticoïde de synthèse (la dexaméthasone) sur les actions biologiques du glucose et du GLP-1.Nous montrons qu’une exposition prolongée des cellules beta à la dexaméthasone exerce des effets délétères en inhibant la sécrétion d’insuline en réponse au glucose et l’activation des kinases de survie ERK1/2 (Extracellular Regulated Kinases 1/2). A l’inverse, nous démontrons que l’exposition prolongée des cellules bêta à la dexaméthasone favorise le maintien du récepteur GLP-1 à la membrane plasmique, augmente le couplage du récepteur à la protéine Galpha s, ce qui se traduit par une production de second messager (AMPc) intracellulaire doublée. Malgré une diminution des effets du glucose, la sécrétion d’insuline et l’activation des kinases ERK1/2 en réponse au GLP-1 ne sont pas affectées. Cette étude révèle qu’une exposition chronique des cellules beta aux glucocorticoïdes 1) régule le trafficking du récepteur GLP-1 et favorise son maintien à la surface cellulaire, 2) hypersensibilise la signalisation du récepteur GLP-1 dépendante de la protéine Gαs , et 3) pourrait impacter les effets thérapeutiques des molécules ciblant l’activation du récepteur GLP-1. / Pancreatic beta cells synthesize and secrete insulin, the only hypoglycemic hormone in the body. These cells play a central role in the onset of diabetes. To protect the functional beta-cell mass is essential. The GLP-1 receptor, which belongs to the class B of the G protein-coupled receptor (GPCR) family, is a major therapeutic target in type 2 diabetes. Through its receptor, GLP-1 potentiates glucose-induced insulin secretion and improves the survival of pancreatic beta cells. Glucocorticoids are stress hormones implied in energetic metabolism and are widely used in therapeutics for their anti-inflammatory, immunosupressive and anti-allergic properties. Neverless, on chronic administration, glucocorticoids can induce metabolic syndrome especially due beta cell functional mass impairement. Here, we characterized the impact of a prolonged exposure of pancreatic beta cells to a synthetic glucocorticoid (dexamethasone) on biological actions of glucose and GLP-1.We show that a chronic exposure of beta cells to dexamethasone exerted deleterious effects on glucose-induced insulin secretion and ERK1/2 (Extracelllular Regulated Kinases 1/2) activation. In contrast, we observed that the glucocorticoid treatment increased GLP-1 receptor expression at the plasma membrane and improved the Galpha s protein coupling leading to an enhancement of cAMP production (2 fold increase). Despite the negative impact on glucose effects, glucocorticoids did not impair neither GLP-1-induced insulin secretion nor ERK1/2 activation. This study reveals that a glucocorticoid chronic exposure 1) regulates GLP-1 receptor trafficking and increases its expression to the plasma membrane, 2) causes supersensitization of Gαs-associated signaling, and 3) could impact on therapeutic effects of GLP-1 receptor-based drugs.

Excess food consumption and/or lack of exercise have dramatically contributed to the prevalence of overweight (BMI≥25) and obesity (BMI≥30) in modern society. The obesity epidemic has been linked to the rise in type 2 diabetes. In recent years, evidence has pointed to a close association between mitochondrial dysfunction in white adipose tissue (WAT) and insulin resistance, a key feature of type 2 diabetes. In order to dissect the cause and effect relationship between WAT mitochondrial dysfunction and insulin resistance, we established an in vitro cell line system to investigate this issue. We artificially introduced mitochondrial dysfunction in 3T3-L1 adipocytes by depleting the mitochondrial transcription factor A (Tfam) during adipogenesis, without changing the overall adipocyte differentiation program. We found that these Tfam-depleted 3T3-L1 adipocytes showed symptoms of insulin resistance, evidenced by impaired insulin stimulated GLUT4 translocation and glucose uptake. This result suggested that mitochondrial dysfunction could be a primary contributor to insulin resistance in fat tissue. However, the exact mechanism underlying this finding remains unclear. As part of a comprehensive understanding of insulin signaling in fat cells, we also investigated the involvement of the endosomal protein WDFY2 in the regulation of Akt isoform-specific effect on glucose uptake. In 3T3-L1 adipocytes, both Akt1 and Akt2 isoforms are expressed, but only Akt2 plays an indispensible role in insulin-stimulated GLUT4 translocation and glucose uptake. Previous studies implied that endosomal proteins may take a part in determining Akt substrate specificity. Here we found that WDFY2 preferentially co-localized with Akt2 and that knockdown of WDFY2 inhibited insulin-stimulated glucose uptake in 3T3-L1 adipocytes, suggesting that endosomes are involved in this regulation. The effect of WDFY2 knockdown on insulin-stimulated glucose uptake worked through the down-regulation of Akt2, but not Akt1, protein level. We concluded that, endosomal protein WDFY2, by preferentially interacting with Akt2, regulates insulin signaling in glucose uptake in 3T3-L1 adipocytes. Our findings may help to develop specific therapeutic interventions for treatment of insulin resistance and type 2 diabetes.

Obesity has increased globally in epidemic proportions and as have the associated disorders. Insulin resistance that could further lead to type 2 diabetes is a major obesity associated dysfunction. Studies using insulin resistant mouse models and observations from human subjects exhibiting insulin resistance provide evidence for ectopic lipid deposition in organs like liver, muscle and heart as one of the major risk factors for developing insulin resistance. These observations suggest that deregulated adipose function to sequester and store excess energy as fat, could lead to insulin resistance. Furthermore, several studies have demonstrated adipose tissue dysfunction leading to inflammation and related syndromes. Interestingly, a mouse model with transgenic expression of glucose transporter in the adipose tissue exhibited improved glucose tolerance and increased insulin sensitivity despite development of obesity, upon high fat feeding. Thus mechanisms that improve adipose function could alleviate insulin resistance and associated diseases. Mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) was identified in our laboratory as a negative regulator of adipocyte function. Interestingly, siRNA mediated knockdown of MAP4K4 promoted PPARγ protein expression. Additionally, silencing of MAP4K4 increased adipocyte triglyceride content. Because MAP4K4 is a negative regulator of PPARγ expression and adipocyte function, understanding the mechanism by which MAP4K4 regulates PPARγ expression is of interest. Thus, for the first part of this thesis, I characterized the signaling pathways utilized by MAP4K4 to regulate PPARγ expression in cultured adipocytes. Here I show that MAP4K4 regulates PPARγ expression through regulation of its protein translation. siRNA mediated MAP4K4 gene silencing stimulated PPARγ protein synthesis without changing its mRNA transcription or its protein degradation. This increase in PPARγ protein translation was due to an increase in the activity of mammalian target of rapamycin (mTOR). The increase in PPARγ protein expression mediated by mTOR activation was a specific effect of the 4E-BP1 phosphorylation that leads to its inactivation and was not a general increase in mTOR activity towards all of its substrates. Finally, adenovirus mediated over expression of MAP4K4 inhibited mTOR activation, and suppressed PPARγ protein translation. For the second part of this thesis, I assessed the role of MAP4K4 in adipocytes in vivo. To accomplish this, a lentivirus mediated shRNA construct was generated to attenuate MAP4K4 expression selectively in the mouse adipose tissue. First we demonstrate that the MAP4K4 shRNA construct is able to efficiently silence the expression of MAP4K4 in vitro when co-expressed with Cre recombinase. Furthermore, we show that following modification of the lentiviral conditional vector that was introduced into a mouse embryo at one cell stage, and crossing the resulting founders with aP2-Cre mice, adipose tissue specific MAP4K4 gene silencing was achieved. Moreover, shRNA mediated gene silencing is a faster and an inexpensive means of achieving tissue specific gene knockdown relative to the available traditional gene knockout approaches. Utilizing these adipose specific MAP4K4 gene knockdown mice, I reveal that MAP4K4 silencing enhanced fat mass as well as PPARγ expression significantly. This is accompanied by improved whole body insulin sensitivity. Furthermore, when challenged with high fat diet, adipose-specific MAP4K4 silenced mice exhibit enhanced adiposity with decreased lean mass. Moreover, adipocyte cell size and triglyceride content are significantly increased. Interestingly, despite increased adiposity, hepatic insulin sensitivity is significantly improved leading to decreased glucose output. Thus MAP4K4 is an important regulator of adipocyte function that mediates whole body glucose homeostasis, through a mechanism that is yet to be identified.

Innate immunity is the first line of defense against invading pathogens. It functions to eliminate pathogens and also to control infections. The innate immune response is also important for the development of pathogen-specific adaptive immune responses. As a result, the study of innate immune signaling pathways is crucial for understanding the interactions between host and pathogen. Unlike mammals, insects lack a classical adaptive immune response and rely mostly on innate immune responses. Innate immune mechanisms have been widely studied in the fruit fly, Drosophila melanogaster. The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophila show strong homology to those of vertebrates making them ideal for studying these pathways. Drosophila immunity relies on cellular and humoral innate immune responses to fight pathogens. The hallmark of the Drosophilahumoral immune response is the rapid induction of antimicrobial peptide genes in the fat body. The production of these antimicrobial peptides is regulated by two immune signaling pathways-Toll and Immune Deficency (IMD) pathways. The Toll pathway responds to many Gram-positive bacterial and fungal infections , while the IMD pathway is potently activated by DAP-type peptidoglycan (PGN) from Gram-negative bacteria and certain Gram-positive bacteria. Two receptors, PGRP-LC and PGRP-LE, are able to recognize DAP-type PGN at the cell surface or in the cytosol, respectively, and trigger the IMD pathway. Upon binding DAP-type PGN, both PGRP-LC and PGRP-LE dimerize/ multimerize and signal to the downstream components of IMD pathway. It is unclear how the receptor activates its downstream components. My work has focused on understanding the molecular events that take place at the receptors following there activation. In these studies I have identified a common motif in the N-terminal domains of both the receptors, known as the RHIM-like domain. The RHIM-like domain is critical for signaling by either receptor, but the mechanism(s) involved remain unclear. IMD, a downstream component of the pathway, associates with both PGRP-LC and -LE but the interaction of PGRP-LC with IMD is not mediated through its RHIM-like domain. Also, mutations affecting the PGRP-LC RHIM-like motif are defective in all known downstream signaling events. However, the RHIM-like mutant receptors are capable of serving as a platform for the assembly of all known components of a receptor proximal signaling complex. These results suggest that another, unidentified component of the IMD signaling pathway may function to mediate interaction with the RHIM-like motif. I performed a yeast two-hybrid screen to identify proteins that might interact with the receptor PGRP-LC through its RHIM- like domain. With this approach, two new components of the IMD pathway were identified. The first component I characterized is called Rudra and it is a critical feedback inhibitor of peptidoglycan receptor signaling. The other factor is known as RYBP, it includes a highly conserved ubiquitin binding motif (NZF), and RNAi studies suggest it is a critical component of the IMD pathway. The identification and characterization of these two new components of the IMD pathway has provided a new insight into the molecular events that take place proximal to the receptor.

Systemic glucose regulation is essential for human survival as low or chronically high glucose levels can be detrimental to the health of an individual. Glucose levels are highly regulated via inter-organ communication networks that alter metabolic function to maintain euglycemia. For example, when nutrient levels are low, pancreatic α-cells secrete glucagon, which signals to the liver to promote glycogen breakdown and glucose production. In times of excess nutrient intake, pancreatic β-cells release insulin. Insulin signals to the liver to suppress hepatic glucose production, and signals to the adipose tissue and the skeletal muscle to take up excess glucose via insulin-regulated glucose transporters. Defects in this inter-organ communication network including insulin resistance can result in glucose deregulation and ultimately the onset of type-2 diabetes (T2D). To identify novel regulators of insulin-mediated glucose transport, our laboratory performed an siRNA-mediated gene-silencing screen in cultured adipocytes and measured insulin-mediated glucose transport. Gene silencing of Mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a Sterile-20-related serine/threonine protein kinase, enhanced insulin-stimulated glucose transport, suggesting Map4k4 inhibits insulin action and glucose transport. Thus, for the first part of my thesis, I explore the role of Map4k4 in cultured adipose cells and show that Map4k4 also represses lipid synthesis independent of its effects on glucose transport. Map4k4 inhibits lipid synthesis in a Mechanistic target of rapamycin complex 1 (mTORC1)- and Sterol regulatory element-binding transcription factor 1 (Srebp-1)-dependent mechanism and not via a c-Jun NH2-terminal kinase (Jnk)-dependent mechanism. For the second part of my thesis, I explore the metabolic function of Map4k4 in vivo. Using mice with loxP sites flanking the Map4k4 allele and a ubiquitously expressed tamoxifen-activated Cre, we inducibly ablated Map4k4 expression in adult mice and found significant improvements in metabolic health indicated by improved fasting glucose and whole-body insulin action. To assess the role of Map4k4 in specific metabolic tissues responsible for systemic glucose regulation, we employed tissue-specific knockout mice to deplete Map4k4 in adipose tissue using an adiponectin-cre transgene, liver using an albumin-cre transgene, and skeletal muscle using a Myf5-cre transgene. Ablation of Map4k4 expression in adipose tissue or liver had no impact on whole body glucose homeostasis or insulin resistance. However, we surprisingly found that Map4k4 depletion in Myf5-positive tissues, which include skeletal muscles, largely recapitulates the metabolic phenotypes observed in systemic Map4k4 knockout mice, restoring obesity-induced glucose intolerance and insulin resistance. Furthermore these metabolic changes were associated with enhanced insulin signaling to Akt in the visceral adipose tissue, a tissue that is nearly devoid of Myf5-positive cells and does not display changes in Map4k4 expression. Thus, these results indicate that Map4k4 in Myf5-positive cells, most likely skeletal muscle cells, inhibits whole-body insulin action and these effects may be mediated via an indirect effect on the visceral adipose tissue. The results presented here provide evidence for Map4k4 as a potential therapeutic target for the treatment of insulin resistance and T2D.

Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.

Human obesity is increasing globally at an impressive rate. The rise in obesity has led to an increase in diseases associated with obesity, such as type 2 diabetes. A major prerequisite for this disease is the development of insulin resistance in the muscle and adipose tissues. Interestingly, experiments in rodent models suggest that adipocytes and macrophages can profoundly influence the development of insulin resistance. Accordingly, the number of adipose tissue macrophages increases substantially during the development of obesity. Numerous research models have demonstrated that macrophages promote insulin resistance by secreting cytokines, like TNFα, which impair whole body insulin sensitivity and adipose tissue function. Additionally, enhancements of murine adipose function, particularly glucose disposal, prevent the development of insulin resistance in mice on a high fat diet. Thus, mechanisms which enhance adipose function or attenuate macrophage inflammation are of interest. Our lab previously identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) as a potent negative regulator of adipocyte function. In these studies, TNFα treatment increased the expression of adipocyte MAP4K4. Furthermore, the use of small interfering RNAs (siRNA) to block the increase in MAP4K4 expression protected adipocytes from some of the adverse effects of TNFα. Because MAP4K4 is a potent negative regulator of adipocyte function, an understanding of the mechanisms by which TNFα regulates MAP4K4 expression is of interest. Thus, for the first part of this thesis, I characterized the signaling pathways utilized by TNFα to regulate MAP4K4 expression in cultured adipocytes. Here I show that TNFα increases MAP4K4 expression through a pathway requiring the transcription factors activating transcription factor 2 (ATF2) and the JUN oncogene (cJUN). Through TNFα receptor 1 (TNFR1), but not TNFR2, TNFα increases MAP4K4 expression. This increase is highly specific to TNFα, as the inflammatory agents IL-1β, IL-6 and LPS did not affect MAP4K4 expression. In agreement, the activation of cJUN and ATF2 by TNFα is sustained over a longer period of time than by IL-1β in adipocytes. Finally, MAP4K4 is unique as the expression of other MAP kinases tested fails to change substantially with TNFα treatment. For the second part of this thesis, I assessed the role of MAP4K4 in macrophage inflammation in vitro and in vivo. To accomplish this task, pure β1,3-D-glucan shells were used to encapsulate siRNA. Glucan shells were utilized because they are effectively taken up by macrophages which express the dectin-1 receptor and they survive oral delivery. I demonstrate that these β1,3-D-glucan encapsulated RNAi particles (GeRPs) are efficiently phagocytosed and capable of mediating the silencing of multiple macrophage genes in vitro and in vivo. Importantly, oral treatment of mice with GeRPs fails to increase plasma IFNγ and TNFα or alter serum AST and ALT levels. Orally administered GeRPs are found in macrophages isolated from the spleen, liver, lung and peritoneal cavity and mediate macrophage gene silencing in these tissues. Utilizing this technology, I reveal that MAP4K4 augments the expression of TNFα in macrophages following LPS treatment. Oral delivery of MAP4K4 siRNA in GeRPs silences MAP4K4 expression by 70% and reduces basal TNFα and IL-1β expression significantly. The depletion of MAP4K4 in macrophages protects 40% of mice from death in the LPS/D- galactosamine (D-GalN) model of septicemia, compared to less than 10% in the control groups. This protection associates with significant decreases in serum TNFα concentrations following LPS/D-GalN challenge. Consistent with reduced macrophage inflammation, hepatocytes from mice treated orally with GeRPs targeting MAP4K4 present less apoptosis following LPS/D-GalN treatment. Thus, MAP4K4 is an important regulator of macrophage TNFα production in response to LPS. The results presented here add to the knowledge of MAP4K4 action in adipocyte and macrophage inflammation substantially. Prior to these studies, the mechanism by which TNFα controlled MAP4K4 expression in adipocytes remained unknown. Considering that MAP4K4 is a negative regulator of adipocyte function, identifying the mechanisms that control MAP4K4 expression was of interest. Furthermore, the role of macrophage MAP4K4 in LPS stimulated TNFα production was also unknown. To address this question in vivo, new technology specifically targeting macrophages was needed. Thus, we developed a technology for non toxic and highly specific macrophage gene silencing in vivo. Considering that macrophages mediate numerous diseases, the application of GeRPs to these disease models is an exciting new possibility.