Immunotherapy targeting isoDGR-protein damage extends lifespan in a mouse model of protein deamidation.

Aging results from the accumulation of molecular damage that impairs normal biochemical processes. We previously reported that age-linked damage to amino acid sequence NGR (Asn-Gly-Arg) results in "gain-of-function" conformational switching to isoDGR (isoAsp-Gly-Arg). This integrin-binding motif activates leukocytes and promotes chronic inflammation, which are characteristic features of age-linked cardiovascular disorders. We now report that anti-isoDGR immunotherapy mitigates lifespan reduction of Pcmt1-/- mouse. We observed extensive accumulation of isoDGR and inflammatory cytokine expression in multiple tissues from Pcmt1-/- and naturally aged WT animals, which could also be induced via injection of isoDGR-modified plasma proteins or synthetic peptides into young WT animals. However, weekly injection of anti-isoDGR mAb (1 mg/kg) was sufficient to significantly reduce isoDGR-protein levels in body tissues, decreased pro-inflammatory cytokine concentrations in blood plasma, improved cognition/coordination metrics, and extended the average lifespan of Pcmt1-/- mice. Mechanistically, isoDGR-mAb mediated immune clearance of damaged isoDGR-proteins via antibody-dependent cellular phagocytosis (ADCP). These results indicate that immunotherapy targeting age-linked protein damage may represent an effective intervention strategy in a range of human degenerative disorders.

Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

Talin1 controls dendritic cell activation by regulating TLR complex assembly and signaling.

Talin critically controls integrin-dependent cell migration, but its regulatory role in skin dendritic cells (DCs) during inflammatory responses has not been investigated. Here, we show that talin1 regulates not only integrin-dependent Langerhans cell (LC) migration, but also MyD88-dependent Toll-like receptor (TLR)-stimulated DC activation. Talin1-deficient LCs failed to exit the epidermis, resulting in reduced LC migration to skin-draining lymph nodes (sdLNs) and defective skin tolerance induction, while talin1-deficient dermal DCs unexpectedly accumulated in the dermis despite their actomyosin-dependent migratory capabilities. Furthermore, talin1-deficient DCs exhibited compromised chemotaxis, NFκB activation, and proinflammatory cytokine production. Mechanistically, talin1 was required for the formation of preassembled TLR complexes in DCs at steady state via direct interaction with MyD88 and PIP5K. Local production of PIP2 by PIP5K then recruited TIRAP to the preassembled complexes, which were required for TLR signalosome assembly during DC activation. Thus, talin1 regulates MyD88-dependent TLR signaling pathways in DCs through a novel mechanism with implications for antimicrobial and inflammatory immune responses.

IRF-7 Mediates Type I IFN Responses in Endotoxin-Challenged Mice.

IRF-7 mediates robust production of type I IFN via MyD88 of the TLR9 pathway in plasmacytoid dendritic cells (pDCs). Previous in vitro studies using bone marrow-derived dendritic cells lacking either Irf7 or Irf3 have demonstrated that only IRF-3 is required for IFN-ß production in the TLR4 pathway. Here, we show that IRF-7 is essential for both type I IFN induction and IL-1ß responses via TLR4 in mice. Mice lacking Irf7 were defective in production of both IFN-ß and IL-1ß, an IFN-ß-induced pro-inflammatory cytokine, after LPS challenge. IFN-ß production in response to LPS was impaired in IRF-7-deficient macrophages, but not dendritic cells. Unlike pDCs, IRF-7 is activated by the TRIF-, but not MyD88-, dependent pathway via TBK-1 in macrophages after LPS stimulation. Like pDCs, resting macrophages constitutively expressed IRF-7 protein. This basal IRF-7 protein was completely abolished in either Ifnar1-/- or Stat1-/- macrophages, which corresponded with the loss of LPS-stimulated IFN-ß induction in these macrophages. These findings demonstrate that macrophage IRF-7 is critical for LPS-induced type I IFN responses, which in turn facilitate IL-1ß production in mice.

Ezh2 Controls Skin Tolerance through Distinct Mechanisms in Different Subsets of Skin Dendritic Cells.

Ezh2, a well-established epigenetic repressor, can down-regulate leukocyte inflammatory responses, but its role in cutaneous health remains elusive. Here we demonstrate that Ezh2 controls cutaneous tolerance by regulating Langerhans cell (LC) transmigration across the epidermal basement membrane directly via Talin1 methylation. Ezh2 deficiency impaired disassembly of adhesion structures in LCs, leading to their defective integrin-dependent emigration from the epidermis and failure in tolerance induction. Moreover, mobilization of Ezh2-deficient Langerin- dermal dendritic cells (dDCs) via high-dose treatment with a weak allergen restored tolerance, which is associated with an increased tolerogenic potential of Langerin- dDCs likely due to epigenetic de-repression of Aldh in the absence of Ezh2. Our data reveal novel roles for Ezh2 in governing LC- and dDC-mediated host protection against cutaneous allergen via distinct mechanisms.

Talin1 Methylation Is Required for Neutrophil Infiltration and Lipopolysaccharide-Induced Lethality.

Talin1, a well-established integrin coactivator, is critical for the transmigration of neutrophils across the vascular endothelium into various organs and the peritoneal cavity during inflammation. Several posttranslational modifications of talin1 have been proposed to play a role in this process. In this study, we show that trimethylation of talin1 at Lys2454 by cytosolic Ezh2 is substantially increased in murine peritoneal neutrophils upon induction of peritonitis. By reconstituting talin1-deficient mouse myeloid cells with wild-type, methyl-mimicking, or unmethylatable talin1 variants, we demonstrate that methylation of talin1 at Lys2454 is important for integrin-dependent neutrophil infiltration into the peritoneal cavity. Furthermore, we show that treatment with an Ezh2 inhibitor or reconstitution of talin1-deficient myeloid cells with unmethylatable talin1 significantly reduces the number of organ-infiltrating neutrophils and protects mice from LPS-induced mortality.

Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.

Estrogen reprograms the activity of neutrophils to foster protumoral microenvironment during mammary involution.

Epidemiological studies have indicated increased risk for breast cancer within 10 years of childbirth. Acute inflammation during mammary involution has been suggested to promote this parity-associated breast cancer. We report here that estrogen exacerbates mammary inflammation during involution. Microarray analysis shows that estrogen induces an extensive proinflammatory gene signature in the involuting mammary tissue. This is associated with estrogen-induced neutrophil infiltration. Furthermore, estrogen induces the expression of protumoral cytokines/chemokines, COX-2 and tissue-remodeling enzymes in isolated mammary neutrophils and systemic neutrophil depletion abolished estrogen-induced expression of these genes in mammary tissue. More interestingly, neutrophil depletion diminished estrogen-induced growth of ERα-negative mammary tumor 4T1 in Balb/c mice. These findings highlight a novel aspect of estrogen action that reprograms the activity of neutrophils to create a pro-tumoral microenvironment during mammary involution. This effect on the microenvironment would conceivably aggravate its known neoplastic effect on mammary epithelial cells.

Isolation and Activation of Murine Lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Post-translational modification-regulated leukocyte adhesion and migration.

Leukocytes undergo frequent phenotypic changes and rapidly infiltrate peripheral and lymphoid tissues in order to carry out immune responses. The recruitment of circulating leukocytes into inflamed tissues depends on integrin-mediated tethering and rolling of these cells on the vascular endothelium, followed by transmigration into the tissues. This dynamic process of migration requires the coordination of large numbers of cytosolic and transmembrane proteins whose functional activities are typically regulated by post-translational modifications (PTMs). Our recent studies have shown that the lysine methyltransferase, Ezh2, critically regulates integrin signalling and governs the adhesion dynamics of leukocytes via direct methylation of talin, a key molecule that controls these processes by linking integrins to the actin cytoskeleton. In this review, we will discuss the various modes of leukocyte migration and examine how PTMs of cytoskeletal/adhesion associated proteins play fundamental roles in the dynamic regulation of leukocyte migration. Furthermore, we will discuss molecular details of the adhesion dynamics controlled by Ezh2-mediated talin methylation and the potential implications of this novel regulatory mechanism for leukocyte migration, immune responses, and pathogenic processes, such as allergic contact dermatitis and tumorigenesis.

The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin.

A cytosolic role for the histone methyltransferase Ezh2 in regulating lymphocyte activation has been suggested, but the molecular mechanisms underpinning this extranuclear function have remained unclear. Here we found that Ezh2 regulated the integrin signaling and adhesion dynamics of neutrophils and dendritic cells (DCs). Ezh2 deficiency impaired the integrin-dependent transendothelial migration of innate leukocytes and restricted disease progression in an animal model of multiple sclerosis. Direct methylation of talin, a key regulatory molecule in cell migration, by Ezh2 disrupted the binding of talin to F-actin and thereby promoted the turnover of adhesion structures. This regulatory effect was abolished by targeted disruption of the interactions of Ezh2 with the cytoskeletal-reorganization effector Vav1. Our studies reveal an unforeseen extranuclear function for Ezh2 in regulating adhesion dynamics, with implications for leukocyte migration, immune responses and potentially pathogenic processes.

c-Rel regulates Ezh2 expression in activated lymphocytes and malignant lymphoid cells.

The polycomb group protein Ezh2 is a histone methyltransferase that modifies chromatin structure to alter gene expression during embryonic development, lymphocyte activation, and tumorigenesis. The mechanism by which Ezh2 expression is regulated is not well defined. In the current study, we report that c-Rel is a critical activator of Ezh2 transcription in lymphoid cells. In activated primary murine B and T cells, plus human leukemia and multiple myeloma cell lines, recruitment of c-Rel to the first intron of the Ezh2 locus promoted Ezh2 mRNA expression. This up-regulation was abolished in activated c-Rel-deficient lymphocytes and by c-Rel knockdown in Jurkat T cells. Treatment of malignant cells with the c-Rel inhibitor pentoxifylline not only reduced c-Rel nuclear translocation and Ezh2 expression, but also enhanced their sensitivity to the Ezh2-specific drug, GSK126 through increased growth inhibition and cell death. In summary, our demonstration that c-Rel regulates Ezh2 expression in lymphocytes and malignant lymphoid cells reveals a novel transcriptional network in transformed lymphoid cells expressing high levels of Ezh2 that provides a molecular justification for combinatorial drug therapy.

MicroRNA miR-124 controls the choice between neuronal and astrocyte differentiation by fine-tuning Ezh2 expression.

Polycomb group protein Ezh2 is a histone H3 Lys-27 histone methyltransferase orchestrating an extensive epigenetic regulatory program. Several nervous system-specific genes are known to be repressed by Ezh2 in stem cells and derepressed during neuronal differentiation. However, the molecular mechanisms underlying this regulation remain poorly understood. Here we show that Ezh2 levels are dampened during neuronal differentiation by brain-enriched microRNA miR-124. Expression of miR-124 in a neuroblastoma cells line was sufficient to up-regulate a significant fraction of nervous system-specific Ezh2 target genes. On the other hand, naturally elevated expression of miR-124 in embryonic carcinoma cells undergoing neuronal differentiation correlated with down-regulation of Ezh2 levels. Importantly, overexpression of Ezh2 mRNA with a 3'-untranslated region (3'-UTR) lacking a functional miR-124 binding site, but not with the wild-type Ezh2 3'-UTR, hampered neuronal and promoted astrocyte-specific differentiation in P19 and embryonic mouse neural stem cells. Overall, our results uncover a molecular mechanism that allows miR-124 to balance the choice between alternative differentiation possibilities through fine-tuning the expression of a critical epigenetic regulator.

Germinal center dysregulation by histone methyltransferase EZH2 promotes lymphomagenesis.

Protection against deadly pathogens requires the production of high-affinity antibodies by B cells, which are generated in germinal centers (GCs). Alteration of the GC developmental program is common in many B cell malignancies. Identification of regulators of the GC response is crucial to develop targeted therapies for GC B cell dysfunctions, including lymphomas. The histone H3 lysine 27 methyltransferase enhancer of zeste homolog 2 (EZH2) is highly expressed in GC B cells and is often constitutively activated in GC-derived non-Hodgkin lymphomas (NHLs). The function of EZH2 in GC B cells remains largely unknown. Herein, we show that Ezh2 inactivation in mouse GC B cells caused profound impairment of GC responses, memory B cell formation, and humoral immunity. EZH2 protected GC B cells against activation-induced cytidine deaminase (AID) mutagenesis, facilitated cell cycle progression, and silenced plasma cell determinant and tumor suppressor B-lymphocyte-induced maturation protein 1 (BLIMP1). EZH2 inhibition in NHL cells induced BLIMP1, which impaired tumor growth. In conclusion, EZH2 sustains AID function and prevents terminal differentiation of GC B cells, which allows antibody diversification and affinity maturation. Dysregulation of the GC reaction by constitutively active EZH2 facilitates lymphomagenesis and identifies EZH2 as a possible therapeutic target in NHL and other GC-derived B cell diseases.

A PRC2-dependent repressive role of PRDM14 in human embryonic stem cells and induced pluripotent stem cell reprogramming.

PRDM14 is an important determinant of the human embryonic stem cell (ESC) identity and works in concert with the core ESC regulators to activate pluripotency-associated genes. PRDM14 has been previously reported to exhibit repressive activity in mouse ESCs and primordial germ cells; and while PRDM14 has been implicated to suppress differentiation genes in human ESCs, the exact mechanism of this repressive activity remains unknown. In this study, we provide evidence that PRDM14 is a direct repressor of developmental genes in human ESCs. PRDM14 binds to silenced genes in human ESCs and its global binding profile is enriched for the repressive trimethylation of histone H3 lysine 27 (H3K27me3) modification. Further investigation reveals that PRDM14 interacts directly with the chromatin regulator polycomb repressive complex 2 (PRC2) and PRC2 binding is detected at PRDM14-bound loci in human ESCs. Depletion of PRDM14 reduces PRC2 binding at these loci and the concomitant reduction of H3K27me3 modification. Using reporter assays, we demonstrate that gene loci bound by PRDM14 exhibit repressive activity that is dependent on both PRDM14 and PRC2. In reprogramming human fibroblasts into induced pluripotent stem cells (iPSCs), ectopically expressed PRDM14 can repress these developmental genes in fibroblasts. In addition, we show that PRDM14 recruits PRC2 to repress a key mesenchymal gene ZEB1, which enhances mesenchymal-to-epithelial transition in the initiation event of iPSC reprogramming. In summary, our study reveals a repressive role of PRDM14 in the maintenance and induction of pluripotency and identifies PRDM14 as a new regulator of PRC2.

Ezh2 regulates anteroposterior axis specification and proximodistal axis elongation in the developing limb.

Specification and determination (commitment) of positional identities precedes overt pattern formation during development. In the limb bud, it is clear that the anteroposterior axis is specified at a very early stage and is prepatterned by the mutually antagonistic interaction between Gli3 and Hand2. There is also evidence that the proximodistal axis is specified early and determined progressively. Little is known about upstream regulators of these processes or how epigenetic modifiers influence axis formation. Using conditional mutagenesis at different time points, we show that the histone methyltransferase Ezh2 is an upstream regulator of anteroposterior prepattern at an early stage. Mutants exhibit posteriorised limb bud identity. During later limb bud stages, Ezh2 is essential for cell survival and proximodistal segment elongation. Ezh2 maintains the late phase of Hox gene expression and cell transposition experiments suggest that it regulates the plasticity with which cells respond to instructive positional cues.

Histone H3K27 methyltransferase Ezh2 represses Wnt genes to facilitate adipogenesis.

Wnt/beta-catenin signaling inhibits adipogenesis. Genome-wide profiling studies have revealed the enrichment of histone H3K27 methyltransferase Ezh2 on Wnt genes. However, the functional significance of such a direct link between the two types of developmental regulators in mammalian cells, and the role of Ezh2 in adipogenesis, remain unclear. Here we show Ezh2 and its H3K27 methyltransferase activity are required for adipogenesis. Ezh2 directly represses Wnt1, -6, -10a, and -10b genes in preadipocytes and during adipogenesis. Deletion of Ezh2 eliminates H3K27me3 on Wnt promoters and derepresses Wnt expression, which leads to activation of Wnt/beta-catenin signaling and inhibition of adipogenesis. Ectopic expression of the wild-type (WT) Ezh2, but not the enzymatically inactive F667I mutant, prevents the loss of H3K27me3 and the defects in adipogenesis in Ezh2(-/-) preadipocytes. The adipogenesis defects in Ezh2(-/-) cells can be rescued by expression of adipogenic transcription factors PPARgamma, C/EBPalpha, or inhibitors of Wnt/beta-catenin signaling. Interestingly, Ezh2(-/-) cells show marked increase of H3K27 acetylation globally as well as on Wnt promoters. These results indicate that H3K27 methyltransferase Ezh2 directly represses Wnt genes to facilitate adipogenesis and suggest that acetylation and trimethylation on H3K27 play opposing roles in regulating Wnt expression.

Activated dectin-1 localizes to lipid raft microdomains for signaling and activation of phagocytosis and cytokine production in dendritic cells.

Lipid rafts are plasma membrane microdomains that are enriched in cholesterol, glycosphingolipids, and glycosylphosphatidylinositol-anchored proteins and play an important role in the signaling of ITAM-bearing lymphocyte antigen receptors. Dectin-1 is a C-type lectin receptor (CLR) that recognizes beta-glucan in the cell walls of fungi and triggers signal transduction via its cytoplasmic hemi-ITAM. However, it is not known if similar to antigen receptors, Dectin-1 would also signal via lipid rafts and if the integrity of lipid raft microdomains is important for the physiological functions mediated by Dectin-1. We demonstrate here using sucrose gradient ultracentrifugation and confocal microscopy that Dectin-1 translocates to lipid rafts upon stimulation of dendritic cells (DCs) with the yeast derivative zymosan or beta-glucan. In addition, two key signaling molecules, Syk and PLCgamma2 are also recruited to lipid rafts upon the activation of Dectin-1, suggesting that lipid raft microdomains facilitate Dectin-1 signaling. Disruption of lipid raft integrity with the synthetic drug, methyl-beta-cyclodextrin (betamD) leads to reduced intracellular Ca2+ flux and defective Syk and ERK phosphorylation in Dectin-1-activated DCs. Furthermore, betamD-treated DCs have significantly attenuated production of IL-2, IL-10, and TNFalpha upon Dectin-1 engagement, and they also exhibit impaired phagocytosis of zymosan particles. Taken together, the data indicate that Dectin-1 and perhaps also other CLRs are recruited to lipid rafts upon activation and that the integrity of lipid rafts is important for the signaling and cellular functions initiated by this class of innate receptors.

Polycomb protein Ezh2 regulates pancreatic beta-cell Ink4a/Arf expression and regeneration in diabetes mellitus.

Proliferation of pancreatic islet beta cells is an important mechanism for self-renewal and for adaptive islet expansion. Increased expression of the Ink4a/Arf locus, which encodes the cyclin-dependent kinase inhibitor p16(INK4a) and tumor suppressor p19(Arf), limits beta-cell regeneration in aging mice, but the basis of beta-cell Ink4a/Arf regulation is poorly understood. Here we show that Enhancer of zeste homolog 2 (Ezh2), a histone methyltransferase and component of a Polycomb group (PcG) protein complex, represses Ink4a/Arf in islet beta cells. Ezh2 levels decline in aging islet beta cells, and this attrition coincides with reduced histone H3 trimethylation at Ink4a/Arf, and increased levels of p16(INK4a) and p19(Arf). Conditional deletion of beta-cell Ezh2 in juvenile mice also reduced H3 trimethylation at the Ink4a/Arf locus, leading to precocious increases of p16(INK4a) and p19(Arf). These mutant mice had reduced beta-cell proliferation and mass, hypoinsulinemia, and mild diabetes, phenotypes rescued by germline deletion of Ink4a/Arf. beta-Cell destruction with streptozotocin in controls led to increased Ezh2 expression that accompanied adaptive beta-cell proliferation and re-establishment of beta-cell mass; in contrast, mutant mice treated similarly failed to regenerate beta cells, resulting in lethal diabetes. Our discovery of Ezh2-dependent beta-cell proliferation revealed unique epigenetic mechanisms underlying normal beta-cell expansion and beta-cell regenerative failure in diabetes pathogenesis.

Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells.

Although in vitro studies of embryonic stem cells have identified polycomb repressor complexes (PRCs) as key regulators of differentiation, it remains unclear as to how PRC-mediated mechanisms control fates of multipotent progenitors in developing tissues. Here, we show that an essential PRC component, Ezh2, is expressed in epidermal progenitors but diminishes concomitant with embryonic differentiation and with postnatal decline in proliferative activity. We show that Ezh2 controls proliferative potential of basal progenitors by repressing the Ink4A-Ink4B locus and tempers the developmental rate of differentiation by preventing premature recruitment of AP1 transcriptional activator to the structural genes that are required for epidermal differentiation. Together, our studies reveal that PRCs control epigenetic modifications temporally and spatially in tissue-restricted stem cells. They maintain their proliferative potential and globally repressing undesirable differentiation programs while selectively establishing a specific terminal differentiation program in a stepwise fashion.