FINBA CONVOCATORIA DE EMPLEO DE UN CONTRATO … · grupo de Inmunología Traslacional del ISPA-HUCA...

Fundación para la Investigación e Innovación Biosanitaria de Asturias Avda. de Roma, s/n 33011

CIF: G-74361817 Área de Gestión de proyectos Página [1]

FINBA CONVOCATORIA DE EMPLEO DE UN CONTRATO ASOCIADO A LAS AYUDAS A

ORGANISMOS PÚBLICOS DE INVESTIGACIÓN PARA APOYAR LAS ACTIVIDADES QUE

DESARROLLEN SUS GRUPOS DE INVESTIGACIÓN EN EL PRINCIPADO DE ASTURIAS EN EL

PERÍODO2018-220GRUPIN-2018(IDI-2018-144)

Oviedo,18deEnerode2019

Se convocaunaplazadeDoctorendisciplinasdeCienciasde laSaludoBiomedicinaenel

grupode investigaciónde Inmunología Traslacionaldel Institutode Investigación Sanitaria

delPrincipadodeAsturias(ISPA).

ENTIDADESFINANCIADORAS:PCTI2013-2017ConsejeríadeEmpleo, IndustriayTurismodelPrincipado de Asturias (IDI-2018-144), cofinanciación FEDER (Fondo Europeo de DesarrolloRegional) y Fundación para la Investigación y la Innovación Biosanitaria del Principado deAsturias–FINBANUMERODEPUESTOSCONVOCADOS:1FUNCIONESESPECÍFICAS

El candidato/a se incorporará al grupo de “Inmunología Traslacional” del Instituto deInvestigación Biosanitaria del Principado de Asturias (ISPA)-Hospital Universitario Central deAsturias(HUCA)enalgunodelosproyectosdesarrolladosporelgrupo.Elgrupoestácentradoenelestudiodelosmecanismosepigenéticosymolecularesqueregulanlosprocesosinmunese inflamatorios en diversos contextos patológicos (principalmente leucemiamieloide aguda,trasplanteyenfermedadrenal),potenciando latraslacióndeestosconocimientosa laclínicamedianteeldesarrollodetratamientosbasadosenlainmunoterapia.

Para ello es necesario que el candidato posea conocimientos del sistema inmune, y de lastécnicas más novedosas utilizadas para su estudio, así como capacidad de liderazgo yautonomíaparallevaracaboestosestudios.SEOFRECE

Incorporación a un grupo de investigación con una gran solidez y trayectoria científica. Elgrupo de Inmunología Traslacional del ISPA-HUCA está dirigido por el Doctor Carlos LópezLarrea (Catedrático de Inmunología de la Universidad de Oviedo y Jefe del Servicio deInmunologíadelHUCA),yformadoporunequipomultidisciplinardeinvestigadoresbásicosyclínicos con una amplia trayectoria profesional principalmente en las áreas de Inmunología,Nefrología y Onco-hematología. El grupo forma parte de la Red Española de InvestigaciónRenal(REDinREN)yhasidofinanciadodesdehacemásde20añosconnumerososproyectoscompetitivos(europeos,nacionalesINTRASALUDyregionales).Nuestrogruporealizaestudiosbásicosdel sistema inmune, concarácter traslacional, implementando técnicas tantoparaeldiagnósticocomoparaeltratamientomedianteinmunoterapia.



ElgrupopertenecealInstitutodeInvestigaciónBiosanitariadelPrincipadodeAsturias(ISPA),adyacente al nuevoHospital Universitario Central de Asturias, proporcionando un ambienteidealparalainvestigaciónclínica.

Elcandidatotendrálaposibilidaddeoptaracontratosdeinvestigaciónpúblicoscompetitivos(Juan de la Cierva Incorporación, Ramón y Cajal /Miguel Servet) dentro del grupo deInmunologíaTraslacional.

REQUISITOS

-DoctorendisciplinasdeCienciasdelaSaludoBiomedicinaconunaantigüedadmínimade3años.-Acreditaciónparatrabajarconanimalesdeexperimentación(BoC)-Altogradodemotivación,iniciativayautonomíaparaeldesarrollodelalaborinvestigadora-Niveldeinglésescrito,leídoyhabladomedio-alto

MÉRITOSAVALORAR

-Estancia/spost-doctoral/esencentrosdereferenciainternacional-PublicacionesindexadasenScienceCitationIndex(WebofScience)deprimerdecilocuartilcomoautorprincipaloautordecorrespondencia-Experienciaenestudiosepigenéticosyanálisisbioinformáticos-Experienciaentécnicasdebiologíamolecularycelular-Experienciaenmodelosanimales-Sevaloraráelconocimientosobreelsistemainmune,acreditadoenpublicaciones.-Capacidaddeconseguirfinanciaciónpúblicaoprivada,yliderarproyectos-Personaresponsable,concapacidaddetrabajoenequipo(referencias)-Entrevista

CONDICIONESDELCONTRATO

-Modalidadcontractual:contratodeaccesoalSistemaEspañoldeCienciayTecnología(art.22LeydelaCiencia)oContratodeInvestigadorPost-Doctoral,asociadoasubvenciónalgrupodeinvestigación“InmunologíaTraslacional”enelISPAGRUPIN-18expedienteIDI-2018-144

-Duración:1año,prorrogableporunañoadicional,ohasta la finalizaciónde lasubvención,conunajornadalaboralde40horas.Incorporacióninmediata.

-Retribución:26.475€eurosbrutosanualesen14pagas.

NOSERÁTENIDAENCUENTAningunacandidaturaquenopresentedocumentación(copia)acreditativadelcumplimientodelosrequisitos.Ladocumentaciónoriginalserápresentadasóloporloscandidatosseleccionados.



FORMAYPLAZODEPRESENTACIÓNDESOLICITUDES:

Se presentará CV, referencias, y copia de la documentación acreditativa de los requisitosexigidosydelosméritosquedeseaquesevalorenenformatopdf,desdelas00:00del19deEnero de 2019 hasta las 23:59 horas del 6 de Febrero de2019 a la dirección de correoelectró[email protected] conelasunto“EMPGRUPIN-144-Postdoc”.ANUNCIOSDESEGUIMIENTODELPROCESOSELECTIVO:

Finalizadoel plazodepresentaciónde solicitudes, se publicará en el tablónde anuncios delHUCA (www.hca.es) y en la sede WEB de FINBA (https://finba.es/) y del ISPA(www.ispasturias.es/), un listado de los solicitantes admitidos y excluidos en el proceso,expresando las causas de exclusión y otorgando un plazo de cinco días naturales para lasubsanacióndedefectos, en casodeno subsanaciónenel plazoestablecido, se consideraráqueelcandidatodesistedesusolicitud.FinalizadoelprocesodeadmisiónporpartedelTribunal,sepublicaráeneltablóndeanunciosdelHUCAyenlasedewebdeFINBAeISPA,lacalificaciónfinaldelosaspirantesadmitidosporordendepuntuación,otorgandounplazodecincodíasnaturalesparaalegaciones.Trasestacalificaciónypublicacióndeloscriteriosevaluables,ysiemprequesuperenunvalormínimode50puntos,serealizaráunaentrevistaalostres(3)candidatosmejorvalorados.Secontrataráalcandidato/aconlapuntuaciónmásalta.Finalizadoelprocesodeselección,sepublicaráeneltablóndeanunciosdelHUCAyenlasedeWebdeFINBAeISPAlaresoluciónparalacontratación.En funcióndel númerode candidatas/os los trámitesdescritos podrán agregarse enun soloacto de resolución y notificación, amparando en todo caso los plazos de subsanación y/oreclamación.

COMPOSICIÓNDELTRIBUNAL:

Presidente:Dr.CarlosLópezLarrea

Vocales:Dra.BeatrizSuarezAlvarezDr.JoséRamonVidalCastiñeiraDr.RamónM.RodriguezLópezDra.AroaBaragañoRaneros



ANEXO1

VALORACIÓNDELASSOLICITUDES

CriterioPuntuaciónmáxima

Valoración

Estanciaspost-doctoralesencentrosdereferenciainternacional. 5 Valoraciónacriteriodeltribunal

PublicacionesindexadasenScienceCitationIndex(WebofScience)deprimerdecilocuartilcomoautorprincipaloautordecorrespondencia.

15

Seasignarán3puntosporpublicacionesdeprimerdecily1,5puntosporpublicacionesdeprimercuartil

Experienciaenestudiosepigeneticosyanálisisbioinformático. 10 Valoraciónacriteriodeltribunal

Experienciaentécnicasdebiologíamolecularycelular. 10 Valoraciónacriteriodeltribunal

Experienciaenmodelosanimales. 10 ValoraciónacriteriodeltribunalConocimientosobreelsistemainmune,acreditadoenpublicaciones. 10 Valoraciónacriteriodeltribunal

Experienciacontrastadaenlacaptaciónderecursosmediantelaobtencióndeproyectosdeinvestigacióncompetitivos(financiaciónpúblicaoprivad)yliderarproyectos.

5 Valoraciónacriteriodeltribunal

Referenciasacreditando:capacidaddetrabajoenequipoyresponsabilidadanteeltrabajoinvestigador.

5 Valoraciónacriteriodeltribunal

Entrevistapersonal. 30 ValoraciónacriteriodelaComisióndeselección

Review

Therapeutic Epigenetic Reprogramming ofTrained Immunity in Myeloid CellsR.M. Rodriguez,1 B. Suarez-Alvarez,1,* and C. Lopez-Larrea1,2,*

Infiltrating and tissue-resident myeloid cells are essential regulators of innateand adaptive immunity. During inflammation, and in response to microbialproducts, these cells can adapt to microenvironmental conditions and acquirespecialized functions, including phagocytosis and the production of proinflam-matory cytokines. Such myeloid plasticity is driven, in part, by epigeneticdynamics that can sustain stable phenotypes after activation, and whichmay lead to maladaptive cell polarization states associated with inflammationand autoimmunity. Here, we review recent reports describing epigenetic mech-anisms linked to such polarization states and innate immune memory (toleranceand training) in monocyte and macrophage lineages. We discuss how thesemechanisms might be targeted to develop putative immunomodulatory toolsthat might be used to treat a variety of immune-mediated diseases.

Myeloid Plasticity and Epigenetic RegulationIn response to inflammation, and guided by the cytokine milieu and interactions with other cellsor microbial products, resident and newly recruited myeloid cells can acquire specializedfunctions. These can include the production of cytokines and nitric oxide, the acquisition ofphagocytic competence, as well as the induction of angiogenesis and antigen presentation. Asa result, the combination and magnitude of environmental cues can induce different activationstates, contributing to the exacerbation of immune responses, or conversely, to the resolutionof inflammation, and the activation of tissue repair mechanisms. In this context, myeloidplasticity plays an essential role by integrating immunomodulatory signals in different phasesof the immune and inflammatory reactions, as well as during pathological processes associatedwith such responses, as in certain cases of autoimmunity and cancer [1].

The plasticity of these cells derives from their capacity to react and adapt to a large catalog ofenvironmental cues and their ability to retain stable phenotypic changes after exposure to stimuli, as inthecaseof innate immunememory.Nonetheless,manyof thesesignalscanbesimultaneouslypresentin inflamed tissues and it is not clear how signaling is integrated during cell fate decisions, or whethercellscanreversetheirphenotypesinresponsetoconflictingpolarizationcues.Epigeneticmechanisms,such as DNA methylation and histone marking (see Glossary), are critical during differentiation andactivationof immunecells [2–5], indicatingthatchromatincanactasan integrationnodeduringmyeloidcell polarization under physiological conditions, as well as in inflamed/damaged tissue sites [6–8]. As aconsequence, it is conceivable that proinflammatory cytokine production, polarization, tolerance, andinnate immunememorymightbetargetedat theepigenetic level, to treatavarietyofconditions,offeringnew putative therapeutic options. Here, we provide an overview of how epigenetic mechanisms candrive macrophage polarization and innate immune memory, and discuss how these might be targetedin disease states, along with the challenges that must be overcome to implement any of theseepigenetic-based strategies. Finally, we review the most recent findings regarding trained immunity intissue-resident macrophages in the central nervous system (CNS) (microglia) and their involvement in

HighlightsEpigenetic mechanisms control thebalance between a resting state andthe activation of monocytes/macro-phages in the presence of inflamma-tory stimuli. These mechanisms arealso essential to induce and maintainpolarization and innate immune mem-ory after activation.

Epigenetic targeting of these pro-cesses can ameliorate inflammationand tissue damage in experimentalmodels of immune and inflammatorydiseases, including rheumatoid arthri-tis, asthma, encephalomyelitis, chronickidney disease, and cancer.

The epigenetic landscape confers along-lasting immune memory onmicroglia that has been associatedwith the severity of certain neurode-generative and neuroinflammatory dis-eases. Peripheral administration oflow-dose LPS induces long-lastinginnate immune memory in microgliathrough epigenetic mechanisms.

1Translational Immunology Laboratory,Instituto de Investigación Sanitaria delPrincipado de Asturias (ISPA),Hospital Universitario Central deAsturias, 33011 Oviedo, Spain2Immunology Department, HospitalUniversitario Central de Asturias,33011 Oviedo, Spain

*Correspondence:[email protected] (B. Suarez-Alvarez)and [email protected] (C. Lopez-Larrea).

66 Trends in Immunology, January 2019, Vol. 40, No. 1 https://doi.org/10.1016/j.it.2018.11.006© 2018 Published by Elsevier Ltd.

mailto:[email protected]

mailto:[email protected]

https://doi.org/10.1016/j.it.2018.11.006

http://crossmark.crossref.org/dialog/?doi=10.1016/j.it.2018.11.006&domain=pdf

GlossaryBET proteins: bromodomain andextra-terminal motif (BET) proteinsare regulatory proteins that reversiblybind acetylated lysine in histones andother nuclear proteins.Chromatin remodeling: changes ofchromatin architecture due tonucleosome positioning that modifyprotein accessibility andtranscriptional activity.Damage-associated molecularpatterns (DAMPs): endogenousmolecules released from necroticand dying cells that activate theimmune system. They are the mainmediators of sterile inflammation.DNA methylation: covalent additionof a methyl group, usually at thecarbon 5 position of cytosines. It isassociated with reduced transcriptionwhen present in regulatory regionssuch as promoters.DNA methyltransferases(DNMTs): a highly conserved familyof proteins aimed at maintaining(DNMT1) or initiating de novo (DNMT3a and 3b) DNA methylation.Endotoxin tolerance: phenomenonthat describes survival of a lethaldose of bacterial endotoxin when ithas been previously beenadministered at a sublethal dose.Enhancer: small region of DNA thatcan be bound by activating proteins,thereby enhancing the transcriptionalactivity of associated genes.Foam cells: fat-laden macrophageslocated in fatty deposits on bloodvessel walls.b-Glucan: a group of b-D-glucosepolysaccharides present in the cellwall of bacteria and fungi.Histone deacetylases (HDACs):enzymes that remove acetyl groupsfrom the lysine residues of histoneand nonhistone proteins.Histone marking: post-translationalmodifications of histone proteins thatalter chromatin functionality andtranscriptional activity.Lipopolysaccharide (LPS):molecule formed by covalentbonding between a lipid and apolysaccharide. They are often foundin the outer membrane of Gram-negative bacteria.Toll-like receptors (TLRs): familyof transmembrane receptorsexpressed by immune andnonimmune cells that recognizeconserved pathogen-associated

neurological disorders. The studies summarized here highlight the future potential of epigenetictargeting in immune and inflammatory disorders mediated by monocytes/macrophages.

Breaking the Epigenetic Barriers of Monocyte and Macrophage ActivationMacrophages are highly heterogeneous immune cells that arise from myeloid progenitors ofembryonic origin and that infiltrate tissues during development [9]. In mice, in some adulttissues, such as gut or heart, macrophage pools can be maintained by circulating monocytesthat differentiate into macrophage cells after infiltration under steady-state conditions [10].Microbial insults and inflammatory signals can trigger the activation of tissue macrophages andinduce recruitment of monocytes capable of differentiating into macrophage-like cells witheffector functions [10]. To trigger this process, activating signals in the inflammatory microen-vironment must overcome transcriptional barriers that maintain cells in a resting state. Thesebarriers are established at the epigenetic level and prevent activation in the absence of stimulisince many promoter regions of Toll-like receptor (TLR) or interferon (IFN)-response genes inmouse bone-marrow-derived macrophages are enriched in repressive epigenetic marks, suchas methylation of H3K27 and H3K9 [11,12], generally with corepressor complexes that canprevent activation [13]. These complexes include epigenetic enzymes such as the histonedeacetylase (HDAC)3, which induces low acetylation levels in histone H3 in proinflammatorygenes to cause transcriptional silencing [14], or the SMYD5 histone methyltransferase, whichmaintains the repressive mark H4K20me3 in nuclear factor (NF)-kB target genes [15]. Subse-quently, these repressive mechanisms can be eliminated in the presence of stimuli, inducing theaccumulation of activating epigenetic marks in promoters and enhancers. Thus, the histonemethyltransferase MLL1, which catalyzes the addition of methyl groups to lysine 4 in histone H3(H3K4), is required for the expression of NF-kB response genes following stimulation viabacterial lipopolysaccharide (LPS) or via induction with the cytokine tumor necrosis factor(TNF)-a [16,17]. Moreover, such epigenetic activation can be accompanied by chromatinremodeling mediated by the SWI/SNF complex, which increases chromatin accessibility [18].Taken together, the findings of these in vitro studies in mouse bone-marrow-derived macro-phages indicate that epigenetic enzymes recruited to regulatory regions of inflammatory genescan play essential roles during the transition from resting to activated states in myeloid cells.

Epigenetic Control of Macrophage PolarizationEpigenetic reprogramming is a fundamental regulatory process operating during macrophageactivation in response to inflammatory stimuli. Nonetheless, an important question in the field ishow epigenetic programs, once triggered, are fine-tuned during the progression from physio-logical to inflammatory responses, such that macrophages can adapt to changing microenvi-ronmental conditions.

Activated cells were originally classified into two phenotypes with respect to their state ofmacrophage polarization: M1 macrophages – characterized by a proinflammatory phenotype,and M2 macrophages – characterized by immunomodulatory and tissue-repair functions [10].Although this simplified view of macrophage polarization only superficially reflects the phenotypiccomplexity associated with the tissue microenvironment in which many proinflammatory andprohomeostatic signals might be simultaneously present, it has proved to be useful for evaluatingphenotypes acquired during inflammation and disease. In any case, it is important to underscorethat the M1/M2 nomenclature should not be used to classify polarization states, since activatedmacrophages can develop a large array of transcriptional programs in response to the microenvi-ronment conditions and which do not fit well into the M1/M2 paradigm [19]. At the transcriptionallevel, many epigenetic mechanisms (including DNA methylation, histone marking, or miRNAs) can

Trends in Immunology, January 2019, Vol. 40, No. 1 67

contribute to the establishment of persistent polarized phenotypes. The DNA methyltransfer-ase DNMT1 can positively regulate the M1 phenotype of the RAW264.7 macrophage cell line [20],while knockdown of DNMT3b can induce M2 polarization in mouse macrophages in vitro [21]. Inaddition to MLL1 and SMYD5 mentioned above, many other histone-modifying enzymes areinvolved in macrophage activation and polarization. For instance, chromatin immunoprecipitationand knockdown experiments in human and mouse macrophages have shown that the histonedemethylase JMJD3 – which catalyzes the demethylation of the repressive mark H3K27me3 – isimportant for fine-tuning the response to proinflammatory signals such as LPS [22,23] or serumamyloid A, thereby maintaining the expression of proinflammatory cytokines in macrophages [24].Although this result suggests that JMD3 may be involved in M1 polarization, the Jmjd3–Irf4 axishas also been associated with M2 polarization in in vivo response to helminth infection (Nippos-trongylus brasiliensis) and in in vivo mouse skin wound healing experiments [25,26]. HDACs areimportant for preventing myeloid cell activation in the absence of stimuli, but also appear to beessential for the establishment of polarized phenotypes (reviewed in [27]). In any case, it isimportant to point out that HDAC enzymes perform diverse functions during the developmentof proinflammatory and anti-inflammatory phenotypes. For instance, HDAC3 knockout mice areknown to exhibit repressed interleukin (IL)-4 and transforming growth factor (TGF)-b signaling inperitoneal and bone-marrow-derived macrophages [28,29] and HDAC3 is also required for the invitro response to LPS in mouse bone-marrow-derived macrophages [30]. By contrast, HDAC4has been reported to act as a positive regulator of anti-inflammatory polarization by inducing Arg1expression (a characteristic marker for myeloid cells with suppressor activity of host T cellresponses and anti-inflammatory functions) in response to IL-4 signaling during mouse dendriticcell differentiation [31]. Taken together, these findings suggest that the activities of epigeneticenzymes are not restricted to a specific polarization phenotype. Of note, regulatory RNAs are alsoimportant epigenetic mechanisms during macrophage polarization since they can also modulatetranscriptional programs in response to different stimuli (recently reviewed in [32]).

Targeting Epigenetic Control of Polarization in Human DiseaseMany studies have highlighted the central role of epigenetic programming in the polarizationprocess. However, a fundamental question is how this process is associated with humandiseases, and what interventions, if any, can be made at the epigenetic level to controlpathogenesis and/or maintenance of disease states.

Macrophage polarization has been thoroughly studied in cancer, prompted by the close linkbetween inflammation and disease progression. For instance, during the initial development ofsolid tumors (such as colon cancer and melanoma), tumor-associated macrophages(TAMs) often exhibit proinflammatory and antitumor activities [10]. Nonetheless, once cancerprogresses, this phenotype is usually shifted towards M2-like phenotypes with high IL-10production, low antitumor activity and proangiogenic functions [10]. It is surprising that despitethe importance of macrophages in cancer, few epigenetic studies have been conducted.Nonetheless, the HDAC inhibitor vorinostat (SAHA) can reduce macrophage infiltration inbreast tumors developed in PyMT mice [33]. In addition, overexpression of decoy receptor(DcR)3, a member of the TNF receptor superfamily in mouse colon carcinoma-derived cells,can induce M2 polarization in TAMs by a process that can be blocked by HDAC inhibition [34],and epigenetic therapy with the DNMT inhibitor, 5-azacytidine (AZA), can reduce macrophageinfiltration in murine ovarian cancer as well as overall tumor burden [35].

Other pathological conditions can also block macrophage plasticity into maladaptive polariza-tion states. Persistent proinflammatory polarization has been observed in many chronicinflammatory and autoimmune conditions [such as rheumatoid arthritis (RA) and systemic

molecular patterns, which areexpressed by microbial pathogens,and DAMPs.Tumor-associated macrophages(TAMs): infiltrating macrophages insolid tumors. TAMs modulate theimmune response against tumor cellsand also other biological processescritical to tumor progression such asangiogenesis and invasion.

68 Trends in Immunology, January 2019, Vol. 40, No. 1

lupus erythematosus (SLE)], and epigenetic targeting has been studied in animal models, withinteresting results. For instance, HDAC inhibitors have been evaluated in myelin-peptide-induced experimental autoimmune encephalomyelitis (EAE), efficiently limiting the developmentof the autoimmune response, reducing inflammation and axonal damage in the spinal cord [36].Moreover, treatment with pan-HDAC inhibitors in the MRL-lpr/lpr mouse model of SLE hasbeen reported to modify the expression of multiple genes involved in immunopathogenesis,including IL-10 and IFN-g, reducing the associated renal pathology [37,38]. HDAC inhibitorshave also been tested in a murine model of allergen-induced asthma by ovalbumin nebulization,exhibiting a positive reduction of airway inflammation and inflammatory cell count (macro-phages, neutrophils, eosinophils, and lymphocytes) relative to controls [39]. Another study in amouse model of cigarette-smoke-induced airway inflammation demonstrated higher IL-10expression in mouse macrophages and reduced smoke-induced production of proinflamma-tory cytokines (IL-1b and IL-8) relative to control animals upon treatment with a pan-HDACinhibitor (MS-275) [40].

Conversely, the link between macrophage polarization and therapeutic effects of HDACinhibition has been more clearly established in preclinical models of RA than in other autoim-mune disorders. In the mouse collagen antibody-induced arthritis model, treatment with anHDAC1-specific inhibitor NW-21 suppressed osteoclast formation and expression of CCL2and CCL3 in monocytes after ex vivo restimulation with LPS [41]. In addition, in mouse and ratRA models, several HDAC inhibitors (MPT0G009, NW-21, and MS-275) reduced macrophageinfiltration, inflammation, and bone loss relative to those in untreated animals [42,43]. Moreover,recent results with inhibitors of histone acetylation readers, BET proteins (bromodomain andextra-terminal motif proteins), emphasize the therapeutic potential at the histone acetylationlevel. Indeed, the BET inhibitor I-BET151 can suppress TLR-ligand-induced secretion ofinflammatory proteins [matrix metalloproteinase (MMP)1, MMP3, IL-6, and IL-8] and thetranscription of genes induced by TNF-a and IL-1b in RA [44]. Furthermore, relative tountreated controls, the BET inhibitor RVX-297 is able to limit disease development in preclinicalmouse models of RA and EAE by reducing joint inflammation and circulating levels of LPS-induced cytokines (IL-6, IL-17, and IFN-g), as well as reducing the response to inflammatorystimuli in ex vivo treated macrophages, lymphocytes, and fibroblasts [45].

In addition to autoimmunity, macrophages play a critical role in any disease with an inflamma-tory component, and therefore, might be potentially exploited for epigenetic intervention. Inatherosclerosis, for instance, DNMT inhibition by 5-aza-2'-deoxycytidine (decitabine, DAC) canreduce M1 polarization in macrophages infiltrated into atherosclerotic plaques in Ldlr!/! mice,inducing an atheroprotective effect [46]. In a mouse model of obstructive nephropathy inducedby unilateral ureteral obstruction, the intraperitoneal injection of the BET-protein inhibitor JQ1reduced the presence of kidney-infiltrating macrophages and the inflammatory responsemediated by NF-kB relative to untreated animals [47]. Therefore, epigenetic inhibitors canbe used to shift polarity or to prevent myeloid cell activation in a large array of inflammatoryconditions (Table 1). However, it remains unclear to what extent data testing epigeneticinhibitors for myeloid cells in preclinical models will be translatable to human diseases, butthis is an active area of investigation that certainly merits further attention.

Innate Immune Memory in Myeloid CellsThe concept of innate immune memory arises from early observations of immune adaptationfollowing previous insult in organisms without adaptive immunity (plants and invertebrates).These processes were confirmed in humans, in which encounters with pathogens were able tosustain immune changes that modified the response to subsequent infections, resulting in the


potentiation (training) or contraction (tolerance) of the immune reaction during a secondaryresponse (Box 1) [48]. Innate immune memory is associated with persistent changes in thereactivity of myeloid, natural killer, and innate lymphoid cells, which can recognize microbialproducts and other stimuli (including endogenous danger products, such as oxidized low-density lipoprotein (oxLDL)] by germline-encoded receptors and cytokines produced inresponse to these stimuli [48]. Here, we focus on the epigenetic changes induced in monocytesand macrophages after initial stimulation and how these affect subsequent immune responses.

Trained Immunity: Exacerbating Autoimmunity and Chronic InflammationThe first evidence of trained immunity in vertebrates came from early observations that injectionof the fungal product b-glucan protects mice from a subsequent lethal injection of Staphylo-coccus aureus [49]. In humans, epidemiological studies have demonstrated that live attenuatedvaccination can protect against other microbial targets by a process associated with trainedimmunity [50,51]. In human cells, the functional reprogramming after b-glucan exposure istriggered by the dectin-1 receptor and the Raf-1-mediated signaling pathway, which increasesthe expression of activation markers associated with M1 and M2 polarization [52]. Moreover,b-glucan induces sustained changes in histone marking in human monocytes, increasing thetrimethylation of H3K4 (H3K4me3) in many key genes, such as those coding for TNF-a, IL-6,

Table 1. Epigenetic Targeting of Myeloid Cell Activation and Preclinical Rodent Models of Disease

Enzymes Epigenetic mark Drug Preclinical model Process Targeted myeloid cell Refs

Pan-HDAC Histone Ac

Vorinostat RA Activation Macrophages [43]

Vorinostat Cancer Polarization TAMs [33]

Valproate Cancer Polarization TAMs [34]

TSA EAE (MS) ? ? [36]

TSA SLE ? ? [37,38]

MS-275 Smoke-induced airway inflammation Polarization Macrophages [40]

HDAC6 Histone AcBML281 RA Activation Macrophage [43]

Tubastatin A Asthma Activation ? [39]

HDAC1 Histone Ac NW-21 RA Differentiation/Activation

Osteoclast/monocytes [41]

BETs Histone Ac readers

I-BET151 RA Activation Macrophages [44]

RVX-297 RA Activation Macrophages [45]

RVX-297 Encephalomyelitis Activation Macrophages [45]

JQ1 Chronic kidney disease Infiltration Macrophages [47]

DNMTs DNA methylation 5AZAAtherosclerosis Polarization Macrophages [46]

Cancer Infiltration TAMs [35]

EZH2 H3K27me3 GSK126 EAE Activation Microglia [80]

KMTs H3K4me3 MTA

– b-glucan training Monocytes [52]

– BCG training Monocytes [54]

– oxLDL training Monocytes [60]

– Uric acid training Monocytes [62]

G9a H3K9me2 BIX01294 – Alarmin tolerance Monocytes [70]


and IL-18 (Figure 1, Key Figure) [52]. Accordingly, inhibition of histone methyltransferase activityusing 50-deoxy-50-methylthio-adenosine (MTA) inhibits TNF-a production, while an inhibitor ofthe histone demethylase LSD-1 (pargyline) does not produce any significant effect, indicatingthat the training response is dependent on histone methyltransferase activity [52]. Similarresults were obtained in another study, which reported changes in H3K27 acetylation(H3K27Ac) at thousands of promoters and distant regulatory regions in response to b-glucanin human primary monocytes [53]. Moreover, trained immunity induced by b-glucan hasrecently been linked to metabolic activation in human monocytes, including the cholesterolpathway and the intermediate metabolite mevalonate [7] (Box 2). The fact that MTA alsoprevents mevalonate-induced training in monocytes emphasizes the importance of chromatinas an integration element of innate signaling.

Another microbial component that can induce enhanced activation in myeloid cells is bacillusCalmette–Guérin (BCG). BCG is a vaccine widely used against tuberculosis, and humanmonocytes isolated after vaccination have shown enhanced production of proinflammatorycytokines, such as IL-1b and IL-6, and H3K4me3 methylation when stimulated with othermicrobial products [54]. Similar to the findings from b-glucan experiments, histone methyl-transferase inhibition, but not histone demethylation, prevented trained immunity effectively inhuman monocytes primed ex vivo with BCG, indicating that training induced by both stimuli isdependent of histone methyltransferase activity [54]. However, in that study, the effect per-sisted in monocytes isolated from blood for 3 months following vaccination. Although residentmacrophages may have a longer lifespan than circulating monocytes, a recent report indicatesthat blood monocytes do not live longer than 7 days in the bloodstream [55]. This short lifespanraises questions about the mechanism that sustains innate immune memory in these cells. In arecent study, genome-wide analysis of histone marking showed that BCG injection in miceinduced persistent epigenetic changes in bone marrow hematopoietic stem cells (HSCs) [56].Therefore, a microbial product can epigenetically modify progenitor and mature cells, whichmay help explain its long-lasting effects in circulating monocytes. However, it is not entirely clearwhether endogenous danger ligands such as lipoprotein A or oxLDL can also reach the bonemarrow and induce sustained epigenetic changes in maladaptive inflammatory situations. This

Box 1. Confusing Terminology of Myeloid Cell ActivationThe interest in innate immune memory mediated by monocytes and macrophages has burgeoned in recent years due toits enormous clinical potential in many human diseases. However, the sheer quantity of published reports has given riseto a terminology that is confusing or even partially redundant. Upon stimulation, activated cells acquire effectorphenotypes. This process is often called polarization, since it implies the acquisition of proinflammatory (M1 polarizationor classical activation) or prohomeostatic (M2 polarization or alternative activation) activation states. Based on thissimplified paradigm and in vitro experimentation, activating signals have been classified as pro-M1 [IFN-g, LPS, TNF-a,granulocyte–macrophage (GM), colony-stimulating factor (CSF), etc.] or pro-M2 inducers [IL-10, TGF-b, IL-13,macrophage colony-stimulating factor (M-CSF), IL-4, etc.] [10]. Accordingly, polarization is considered a differentiationprocess, since it involves the acquisition of mature and stable phenotypes. On the one hand, the term plasticity indicatesthe ability of these cells to acquire multiple phenotypes in response to different stimuli, but also the capacity to modifytheir phenotypes after activation and in response to changes in the tissue microenvironment, for example, from M1 toM2 signatures. Trained immunity, on the other hand, describes the process by which a primary stimulus (or priming)enhances the activating response to a subsequent microbial signal [48]. Since primary and secondary stimuli are usuallydifferent, this process is considered a cross-protective immune response. Tolerance would be the opposite process, bywhich priming induces a diminished response after a secondary stimulus. When primary and secondary stimuli aredifferent, (e.g., LPS tolerance induced by TNF priming), this process is called cross-tolerance [68]. Overall, training andtolerance are usually terms applied to innate memory-induced responses in vivo, while polarization most often indicatesthe differentiation process that leads to sustained activation states associated with inflammation, tissue damage, anddisease. It should be noted, however, that polarization also involves the acquisition of persistent phenotypes that arelikely to create quantitative or qualitative differences upon exposure to secondary stimuli and, consequently, are alsohighly relevant in the context of innate immune memory.


Key Figure

Epigenetic Dynamics Associated with Trained Immunity and Tolerance

Training

Sustained LPS sƟmulus

SubsequentsƟmulus

Transient acƟvaƟon

NontoleranceTolerance

AcƟvated and primed

FirstsƟmulus

LPS

`-glucan, BCGoxLDL, uric acid

ResƟng

H3K27me H3K9me3

HDAC3 SMYD5Gene

H3K4me1

Gene

H3K27 Ac

HMTs

H3K4me3

Gene

H3K27 Ac

NF-gB

SWI/SNF

IL-6

IL-1`

TNF-_

`-glucan+ LPS

Frp1Lcn2Oasl1

H3K27 Ac

GeneNF-gB

SWI/SNF

miRs

Gene

H3K27 AcH3K4me3

TFs

H3K9me3

G9aHP1

Gene

H3K9me3

NCOR

Il1bIl6Mmp13

NF-gB

Il1bIl6Tnfa

Figure 1.

(Figure legend continued on the bottom of the next page.)

Training immunity is closely associated with epigenetic reprogramming. An initial microbial product, such as b-glucan, induces activation and epigeneticreprogramming in the cell. After elimination of the stimulus, resting cells partially retain their activating epigenetic marks in regulatory regions (promoters and enhancers)associated with immune response genes. This primed state allows strong stimulation in response to a subsequent stimulus, which is also associated with the gain ofactivating marks and chromatin remodeling. In the model of endotoxin-induced tolerant, primary stimulation with LPS induces transient activation of the transcriptional


is likely, since it is well known that pathological chronic inflammation (as in RA) can inducechanges in the differentiation potential of murine HSCs; however, this has not yet been studiedat the epigenetic level, and thus, further studies are clearly warranted [57,58]. If endogenousligands can induce epigenetic changes in the bone marrow, it will then be critical to study theeffect of epigenetic inhibition in HSCs using in vivo models of disease, assessing long-termoutcome in these populations.

Overall, epigenetic regulation seems to play a critical role during trained immunity. Nonetheless,one important question is how this mechanism is associated with maladaptive states in humanpathology and whether epigenetic targeting is a viable therapy [59]. Indeed, several endoge-nous signals associated with inflammation and disease have been linked to trained immunity. Inatherosclerosis, monocytes recruited to the arterial wall produce proinflammatory cytokines.These recruited monocytes are also able to engulf oxLDL, generating lipid-filled foam cells inthe vascular wall, inducing a trained phenotype via the TLR pathway and epigenetic reprog-ramming (enrichment of H3K4me3 on gene promoters coding for TNF-a, IL-6, or IL-18), asevidenced from chromatin immunoprecipitation studies in oxLDL-primed human monocytes[60]. Moreover, pretreatment of monocytes with a histone methyltransferase inhibitor 50-deoxy-50-methylthio-adenosine (MTA) prevented oxLDL-trained production of IL-6 and TNF-a after invitro restimulation with LPS, similar to what has been observed with b-glucan or BCG,

Box 2. Metabolic versus Epigenetic ReprogrammingPolarization, tolerance, and training immunity are processes tightly linked to metabolic changes in myeloid cells. Forinstance, proinflammatory phenotypes in macrophages are associated with increased glycolysis, while homeostaticmacrophages show energy metabolism that is coupled with oxidative phosphorylation and b-oxidation [48]. But, howare the epigenetic and metabolic pathways interconnected? Since the transcriptional program is tightly regulated byepigenetic mechanisms, metabolic pathways can clearly be regulated at this level. Training immunity, for instance,induces epigenetic activation of glycolytic genes, stimulating aerobic glycolysis in myeloid cells [85]. Nonetheless, recentstudies have demonstrated that metabolism can also shape myeloid functions, or even act as a driver of the activationprocess. For instance, blocking glycolysis is certainly enough to inhibit training in human monocytes, increasingH3K4me3 and decreasing H3K9me3 in the IL6 and TNFA gene promoters [86]. Moreover, intermediate metabolitesgenerated during energy metabolism can modify the activity of epigenetic enzymes [87]. Indeed, in human monocytes,the ratio of the tricarboxylic acid (TCA) cycle metabolites a-ketoglutarate and succinate is a critical determinant in theactivity of two families of enzymes controlling epigenetic modifications during macrophage activation, the JMJ (Jumonjidomain-containing) family of lysine demethylases and the TET (ten-eleven translocation) family of methylcytosinehydroxylases involved in DNA demethylation [87].

While it is easy to envision how the availability of an intermediate metabolite could limit the activity of chromatin-modifierenzymes, it is not clear how changes in endogenous levels of these molecular species could induce epigenetic changesin a directed way. A recent report demonstrated that pharmacological inhibition of the cholesterol pathway by statinswas enough to inhibit training in human monocytes [7]. This process appears to be dependent on an intermediatemetabolite in the pathway, mevalonate, that induces training in macrophages via activation of insulin-like growth factor 1(IGF1) receptor and mammalian target of rapamycin (mTOR), in turn leading to epigenetic changes in inflammatorygenes [7]. Thus, the link between metabolism and the epigenome is complex, involving transcriptional regulation ofmetabolic enzymes, intermediate metabolites acting as substrates and cofactors of epigenetic enzymes, as well as thedirect interaction of these molecules with various signaling pathways.

program associated with the immune response in monocytes or macrophages. This process is linked to chromatin remodeling, loss of repressive epigenetic marks, andgain of activating marks. However, sustained stimulation with LPS ultimately leads to a tolerant state in which cells are refractory to further stimulation. At the molecularlevel, this is achieved by recapturing the repressive mechanism at regulatory regions and by the action of miRNAs. Nonetheless, the activation program is not fullysilenced since a significant fraction of genes remain in an activated chromatin state, particularly those associated with antimicrobial functions. The tolerance state can bepartially reversed in response to some stimuli, such as b-glucans [53,56,63]. Abbreviations: BCG, bacillus Calmette–Guérin; HDAC3, histone deacetylase 3; HMT,histone methyltransferase; IL, interleukin; LPS, lipopolysaccharide; miR, miRNA; NF-kB, nuclear factor-kB; oxLDL, oxidized low-density lipoprotein; TNF-a, tumornecrosis factor-a.


indicating that this putative therapeutic strategy might be feasible under other pathologicalconditions in which trained immunity contributes to disease; however, this remains to be tested.In addition, trained immunity has also been associated with gout: priming human monocyteswith uric acid ex vivo can enhance the inflammatory response via epigenetic remodeling, butcan be efficiently prevented by the inhibitor MTA [61,62]. Finally, other endogenous damage-associated molecular patterns (DAMPs) associated with tissue damage, such as purinemetabolites and S100 proteins, may be able to trigger trained immunity during chronicinflammation and autoimmune disorders. However, in vivo studies are needed to assessthe therapeutic potential of epigenetic targeting in this context.

Innate Tolerance: A Strategy for SepsisInitial molecular evidence that myeloid cells may be involved in innate tolerance was obtainedfrom in vitro and in vivo studies in response to LPS in mouse macrophages that showed thatsome genes, such as IL6 or IL1B, can undergo transient silencing after persistent stimulationwith LPS [63]. At the molecular level, LPS tolerance is largely regulated by epigenetic mecha-nisms, including nucleosome remodeling, histone marking, and diminished recruitment oftranscription factors and chromatin remodeling complexes in LPS response genes (Figure 1)in monocytes and macrophages. The molecular mechanisms that drive tolerance are notentirely clear, but NF-kB is known to recruit the NCOR–HDAC3–p50 repressive complex totargeted genes in bone-marrow-derived mouse macrophages [64]. Additionally, the RelBsubunit of NF-kB can recruit the histone H3K9 methyltransferase G9a to promoters, inducingbinding of the heterochromatin protein 1 and epigenetic silencing in THP1 human promon-ocytes [65,66], while nontolerized genes maintain activating chromatin marks and the recruit-ment of chromatin remodeling complexes after secondary LPS stimulation, allowing geneexpression in bone-marrow-derived mouse macrophages [53,63]. In addition, a recent study inthese cells indicated that tolerance could also be regulated at the epigenetic level by miRNAs(miR-221 and miR-222) that induced transcriptional silencing of inflammatory genes viasilencing of the ATP-dependent chromatin remodeler, SMARCA4 [67]. Noteworthy, humanmonocytes primed in vitro with other proinflammatory stimuli, such as TNF, can also stimulateepigenetic silencing of LPS response genes such as IL6, inducing endotoxin tolerance aftersecondary stimulation [68].

Endotoxin tolerance probably evolved as a protective mechanism from the cytokine stormphenomenon associated with sepsis but, in turn, can greatly increase the risk of widespreadinfection due to secondary infection during the tolerant phase [69]. Consequently, rebootinginnate immunity at the epigenetic level might be a promising therapeutic target for improvingsurvival after sepsis. We posit that G9a and HDAC enzymes would be suitable targets sinceboth are involved in silencing NF-kB genes [64–66]. Moreover, in vitro prestimulation of humanmonocytes with alarmins such as MRP8 and MRP14–released during sterile inflammation andassociated with polytrauma and burn trauma – can also induce LPS tolerance by a processdependent on the G9a-repressing activity of NF-kB-targeted genes [70]. By contrast, in vitroHDAC inhibition with Trichostatin A (TSA) in mouse macrophages is able to prevent tolerance(reversing epigenetic silencing of some inflammatory genes, such as IL6) when administeredduring primary or secondary stimulation with LPS [63].

However, sustained LPS stimulation does not necessarily lead to tolerance, since the presenceof some stimuli, such as IFN-g, can prevent it by enhancing the production of proinflammatorycytokines, including IL-6 and TNF-a [71]. This process is mediated by a persistent associationof some transcription factors after IFN-g stimulation, such as signal transducer and activator oftranscription (STAT)1 and IFN-regulatory factor (IRF)-1, at regulatory regions of TLR4


downstream targets that maintain histone acetylation and prevent epigenetic silencing inprimary human macrophages [72]. Yet, an important question remains to be answered: is itpossible to revert tolerance once it has been established? IFN-g treatment of human peripheralblood mononuclear cells can at least restore metabolic features associated with immunopar-alysis in human patients with fungal sepsis [73], suggesting that this may be the case. Of note,Candida albicans b-glucan, which is a potent inducer of trained immunity, can also reverseLPS-induced tolerance in ex vivo-treated human monocytes, rescuing cytokine release andinflammatory phenotype [6]. This process has been linked to the restoration of activationhistone marks such as H3K27Ac. However, in this study, genome-wide epigenetic analysis ofhistone marks demonstrated that restoration was only partial, affecting only 60% of tolerizedgenes [6]. Therefore, targeting epigenetic mechanisms might be a potential alternative orcomplementary strategy to prevent sepsis. Yet, initial experiments with inhibitors of BETproteins have been able to prevent LPS-induced tolerance but not reverse it once it had beenestablished [6]. It would be interesting to test in preclinical models whether other epigeneticinhibitors capable of preventing endotoxin tolerance (such as G9a or HDAC inhibitors) cancompletely reverse it, alone or in the presence of other stimuli, such as b-glucan. In any case,tolerance and training are immune processes clearly associated with an epigenetic rewiring ofthe transcriptional program in myeloid cells, although further experimentation will be necessaryto understand how epigenetic targeting can be achieved to treat such pathologies.

Epigenetic Control of Microglia: A Suitable Target in Neurological DiseasesFrom another angle, microglia – the resident macrophages of the CNS – play a vital role innormal brain function and are major players in innate memory under pathological conditions.Like other macrophage populations, microglia have the ability to respond to peripheral inflam-mation or damage signals by producing proinflammatory cytokines and by inducing recruit-ment of additional immune cells. These cells are essential for maintaining brain homeostasis,although exacerbated activation is also involved in the pathogenesis of neurodegenerative andneuroinflammatory diseases (Alzheimer’s and Parkinson’s diseases, stroke, multiple sclerosis,and dementia) [74]. Microglia arise from a hematopoietic precursor during embryonic devel-opment and further differentiate into mature phenotypes in the CNS [75]. This is orchestrated bykey transcription factors (IRF8, PU.1, SALL 1, and MAFB) and by establishing particularepigenetic modifications in regulatory regions, thereby determining the transcriptional programand function of microglia in healthy or pathological contexts [76].

In the CNS, microglial cells are maintained in a steady state, closely regulated by a subset ofmechanisms, or microglial checkpoints (Figure 2) [77]. One study first demonstrated that dailyintraperitoneal administration of low-dose LPS for four consecutive days induced long-lastinginnate immune memory in mouse microglia [78]. After that, second administration of LPSinduced training, characterized by increased levels of pro- and anti-inflammatory cytokines,whereas sustained stimulation induced a tolerant state, characterized by low level production ofproinflammatory cytokines (IL-1b, TNF-a, and IL-6) and increased IL-10 production (Figure 2)[78]. Accordingly, in an APP23 mouse model of Alzheimer’s disease (AD) pathology, relative tountreated animals, one course of LPS injection (training-inducing stimulus) reduced IL-10production and increased amyloid-b (Ab) levels and plaque load, while four courses of LPSinjections reduced production of IL-1b, IL-6, and IL-12, and ameliorated tissue damage(decreased size and load of Ab plaques) [78]. Studies of chromatin immunoprecipitation formonomethylation at H3K4 (H3K4me1) and H3K27Ac, two markers of active enhancers inmicroglia, revealed that training and tolerance induction with LPS was accompanied byepigenetic changes in enhancers, maintaining cells in a primed state, and allowing fasttranscriptional changes following subsequent LPS stimulation [78]. Trained microglia were


Primed/ resƟng

• Increased Socs3• Increased TRAF6 ubiquiƟnaƟon• Silencing of NF- κB-associated

proinflammatory genes

GSK126

GSK-J4

EZH2

JMJD3

Gene

AcƟve enhancer

H3K27 AcH3K4me1

Steady state

TFG-`

CD200R

IL-4

MeCP2

Gene

(LPS, A`)

IL-1` TNF-_

FirstsƟmulus

ResoluƟon ofinflammaƟon

Second sƟmulus(LPS)

Persistent sƟmulus(LPS)

AcƟvated

Gene

K27

Promoter

NF-gB

IL-10

IL-1`

A` plaques

IL-6 IL-1`

IL-10

A` Plaques

TNF-_

H3K27me3

Gene

Promoter

ToleranceTraining

• Decreased cytokines/chemokines• Decreased IFN-sƟmulated genes• Decreased STAT-1 and IRF7• Blockade of Arg-1 (M2

phenotype)

• Increased H3K4me1 and H3K27Ac in genes related toHIF-1α and inflammatorysignaling (TLR,mTOR,TNF)

• Increased H3K4me1 in genes related to phagocytosis (rap-1 signaling)

Figure 2.

(Figure legend continued on the bottom of the next page.)

Epigenetic Memory as a Therapeutic Target in the Microglial Response. Microglia are maintained in a steady state that is closely controlled byvarious mechanisms (TGF-b levels, CX3CR1–CX3CL1 engagement, transcriptional regulators, such as MeCP2), or microglial checkpoints. Alterations in brainhomeostasis (the result of aging, infection, injury, or disease) or the presence of a stimulus activate the microglia. This process is triggered by epigenetic and molecularmodifications aimed at increasing the expression of inflammatory molecules and enhancing phagocytosis in order to clear the initial stimulus. Activation is highlyregulated by the methylation levels of H3K27 mediated by the EZH2 and JMJD3 enzymes. Pharmacological blockade of both enzymes (GSK126 and GSK-J4) inhibitsthe expression of several genes associated with activation. After activation has taken place, the microglia return to a resting stage, although some epigeneticmodifications (H3K4me1 and H3K27 Ac) remain in latent enhancers, maintaining the ability of long-lasting memory. In experimental rodent models of infection orneurological diseases, exposure to a second (trained) or prolonged (tolerance) LPS stimulation enables the fast activation of enhancers associated with genes involvedin different pathways. Trained immunity is characterized by the activation of HIF-1a and inflammatory pathways, leading to the secretion of elevated levels ofproinflammatory cytokines. Tolerance results in high levels of anti-inflammatory IL-10 cytokine and increased phagocytosis, leading to the ingestion of material such as


characterized by increased H3K4me1 and H3K27Ac markers in active enhancers of thyroidhormone and hypoxia-inducible factor (HIF)-1a signaling pathway genes similar to findings oftrained immunity in macrophages [78]. By contrast, tolerance induction was associated withincreased H3K4me1 marks in putative enhancers of genes related to phagocytic function(Rap1 signaling pathway), potentiating their expression and facilitating the uptake and phago-cytosis of Ab proteins by microglia (Figure 2) [78]. These results were consistent with findings inthe HDAC1/2-deficient AD mouse model, showing reduced Ab plaque loads along withimproved cognitive functions (learning and memory) in the mice relative to wild-type mice [79].

Other epigenetic mechanisms involved in microglia activation have also been implicated in thedevelopment of neuroinflammatory diseases. The specific ablation of Ezh2 (a component of thePCR2 polycomb mediating H3K27me3 repressive mark) in microglia from an experimentalmouse model of autoimmune encephalomyelitis (induced by MOG35–55 and pertussis toxin),reduced disease severity and infiltration of immune cells into the CNS, compared with theirEzh2-sufficient counterparts [80]. Inhibition of Ezh2 reduced H3K27me3 marks in the Socs3gene, allowing Socs3 expression and leading to Lys48 ubiquitination of TNF receptor-associ-ated factor (TRAF)6 and its subsequent degradation by the proteasome (Figure 2). SinceTRAF6 is an essential adaptor of MyD88-dependent NF-kB activation [81], its loss in Ezh2-deficient macrophages led to the silencing of NF-kB-dependent proinflammatory genes.Additionally, treatment of primary and BV-2 microglia cells with the H3K27me3 demethylaseJMJD3 inhibitor, GSK-J4, inhibited the expression of LPS-induced genes (cytokines, chemo-kines, and IFN-stimulated genes) and transcription factors such as STAT1 and IRF7, modulat-ing the inflammatory responses (Figure 2) [82]. However, Jmjd3 can also enhance anti-inflammatory M2 polarization of N9 microglia, effecting a decrease in H3K27me3 and increas-ing Arg1 gene expression, which is relevant as this has been deemed an essential marker of M2polarization and neuronal survival [83]. Moreover, suppression of Jmjd3 in N9 cells leads toenhanced M1 microglia polarization and causes exacerbated dopamine-mediated neuronaldeath in an in vivo mouse model of Parkinsons disease MPTP-intoxication [84].

In summary, epigenetic mechanisms are deemed essential for the polarization, activation, andestablishment of memory in microglia. Epigenetic targeting of these processes might beexploited to slow the progression of neurodegenerative and neuroinflammatory diseases,but this possibility is yet to be empirically explored.

Concluding RemarksEpigenetic dynamics play an essential role in maintaining functional states of myeloid cells andduring innate immune memory associated with infection and chronic inflammation. Conse-quently, epigenetic inhibitors might be used to target activation processes in monocytes andmacrophages associated with human diseases, including microglial cells during CNS pathol-ogy. Although epigenetic targeting is a promising strategy, several aspects must be addressedif a suitable therapy is to be developed. First, epigenetic inhibitors generally have a systemiceffect and so can be extremely unspecific. Also, most epigenetic inhibitors used in preclinicalmodels, such as TSA or SAHA, have a broad range of specificities. These drugs are likely tohave potent biological activity, but selective isoform inhibition is necessary to ensure that theyexert a selective impact on targeted processes while reducing toxicity and adverse effects. This

Clinician’s CornerMonocytes and tissue-resident mac-rophages are critical intermediaries inall human diseases with an inflamma-tory component. They produceimmune mediators (interleukins, che-mokines, leukotrienes, and prosta-glandins, etc.) and stimulate immunecell recruitment and activation.

These cells maintain the balancebetween the pro- and anti-inflamma-tory functions that can be subverted inautoimmune diseases and cancer.This process is regulated at the molec-ular level by epigenetic mechanisms.

The epigenetic landscape establishedin monocytes and macrophages afterinitial activation bestows a stable phe-notype on these cells, allowing rapidgene expression in response to sub-sequent stimuli.

Epigenetic inhibitors can modify thecytokine profile of monocytes andmacrophages or prevent activation inresponse to stimuli. In the context ofcancer, they can be used as comple-mentary immunotherapy becauseenhancing proinflammatory pheno-types can increase immune cell infiltra-tion and improve the immunogenicityof the tumor. On the one hand, sincemany epigenetic inhibitors are alsopotent antitumor drugs, it would beworth investigating whether there areany synergistic effects. In the case ofautoimmunity, on the other hand,these drugs might be used to preventactivation or to shift phenotypestowards anti-inflammatory profiles.

Immunomodulation at the epigeneticlevel could prove to be a powerful toolfor the treatment of immune-relateddiseases. Nonetheless, it should benoted that epigenetic drugs may alterother cellular processes in the inflamedtissue because epigenetic mecha-nisms are not cell specific and are crit-ical transcriptional regulators in mostcellular contexts. To implement thisstrategy it will be essential to evaluate

Ab plaques that are broken down. Microglial training can exacerbate the severity of neuroinflammatory diseases, whereas microglia tolerance might alleviate pathology[78]. Abbreviations: Ab, amyloid b; HIF-1a, hypoxia-inducible factor-1a; IL, interleukin; IRF7, interferon-regulatory factor 7; LPS, lipopolysaccharide; STAT1, signaltransducer and activator of transcription 1; TGF-b, transforming growth factor-b; TRAF6, TNF receptor-associated factor 6.


is especially important since many diseases associated with chronic inflammation require long-term treatment. However, it will be interesting to analyze whether simultaneous inhibition ofdifferent epigenetic mechanisms can improve treatment efficacy or exert synergistic effects. Sofar, there have been no such studies of autoimmune disorders, but the combined inhibition ofDNMT and HDAC did increase M2 polarization in lung tissues in a preclinical mouse model ofendotoxemia-induced acute lung injury [84]. This suggests that combinatorial treatment mightrepresent a suitable strategy to treat certain pathological conditions, but this remains to betested. In any case, epigenetic inhibitors clearly exert an immunomodulatory effect when testedin in vitro models. However, due to the nonspecificity and potential toxicity of this therapeuticstrategy, it is difficult to predict clinical potential (see Outstanding Questions). Preliminaryinvestigations have provided promising results, but further effort is needed to robustly testthese drugs in preclinical models of chronic inflammation and autoimmunity.

AcknowledgmentsThis work was supported by Plan Nacional de I+D+I 2013-2016 and European Union Fondos FEDER, Instituto de Salud

Carlos III [grant number PI16/01318 and PI17/01244] and Red Española de Investigación Renal (REDinREN, grant number

RD16/0009/0020). R.M. Rodriguez is supported by the Ministry of Economy and Competitiveness through a Juan de la

Cierva postdoctoral fellowship (FJCI-2015-24036).

Disclaimer StatementThe authors declare no conflicts of interest.

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Outstanding QuestionsHow do other myeloid cell populations,such as neutrophils or dendritic cells,contribute to the development ofinnate immune memory?

How do different tissue-resident mac-rophages contribute to innate immunememory in response to systemicstimuli?

How can epigenetic targeting be effi-ciently directed against specific cellpopulations?

Can highly specific epigenetic inhibi-tors exert reduced toxicity while con-tinuing to be biologically active?

Can epigenetic drugs induce synergis-tic effects by acting simultaneously ondifferent cell populations andprocesses?

Can metabolic and epigenetic reprog-ramming be targeted simultaneouslyto achieve a more efficient therapeuticstrategy?

What stimuli induce immune tolerancein microglia and could be a therapeuticstrategy in treating neuroinflammatorydiseases?

Are the epigenetic mechanisms thatare important for immune and inflam-matory responses mediated by otherCNS cells, such as astrocytes?

Can the epigenetic profile establishedduring microglia training be affected bygut-brain axis or metabolites derivedfrom gut-dwelling microbes?


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