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1.  Tissue-Resident Macrophage Enhancer Landscapes Are Shaped by the Local Microenvironment 
Cell  2014;159(6):1312-1326.
SUMMARY
Macrophages are critical for innate immune defense and also control organ homeostasis in a tissue-specific manner. They provide a fitting model to study the impact of ontogeny and microenvironment on chromatin state and whether chromatin modifications contribute to macrophage identity. Here, we profile the dynamics of four histone modifications across seven tissue-resident macrophage populations. We identify 12,743 macrophage-specific enhancers and establish that tissue-resident macrophages have distinct enhancer landscapes beyond what can be explained by developmental origin. Combining our enhancer catalog with gene expression profiles and open chromatin regions, we show that a combination of tissue- and lineage-specific transcription factors form the regulatory networks controlling chromatin specification in tissue-resident macrophages. The environment is capable of shaping the chromatin landscape of transplanted bone marrow precursors, and even differentiated macrophages can be reprogramed when transferred into a new microenvironment. These results provide a comprehensive view of macrophage regulatory landscape and highlight the importance of the micro-environment, along with pioneer factors in orchestrating identity and plasticity.
doi:10.1016/j.cell.2014.11.018
PMCID: PMC4437213  PMID: 25480296
2.  Functional classification of memory CD8+ T cells by CX3CR1 expression 
Nature Communications  2015;6:8306.
Localization of memory CD8+ T cells to lymphoid or peripheral tissues is believed to correlate with proliferative capacity or effector function. Here we demonstrate that the fractalkine-receptor/CX3CR1 distinguishes memory CD8+ T cells with cytotoxic effector function from those with proliferative capacity, independent of tissue-homing properties. CX3CR1-based transcriptome and proteome-profiling defines a core signature of memory CD8+ T cells with effector function. We find CD62LhiCX3CR1+ memory T cells that reside within lymph nodes. This population shows distinct migration patterns and positioning in proximity to pathogen entry sites. Virus-specific CX3CR1+ memory CD8+ T cells are scarce during chronic infection in humans and mice but increase when infection is controlled spontaneously or by therapeutic intervention. This CX3CR1-based functional classification will help to resolve the principles of protective CD8+ T-cell memory.
The function of memory CD8+ T cells is often believed to be directly correlated with their localization in tissues. Here the authors show that CD8+ T cells with different proliferative and cytotoxic properties can be distinguished based on their expression of CX3CR1, independently of their tissue localization.
doi:10.1038/ncomms9306
PMCID: PMC4667439  PMID: 26404698
3.  Chromatin state dynamics during blood formation 
Science (New York, N.Y.)  2014;345(6199):943-949.
Chromatin modifications are crucial for development, yet little is known about their dynamics during differentiation. Hematopoiesis provides a well-defined model to study chromatin state dynamics, however technical limitations impede profiling of homogeneous differentiation intermediates. We developed a high sensitivity indexing-first chromatin immunoprecipitation approach (iChIP) to profile the dynamics of four chromatin modifications across 16 stages of hematopoietic differentiation. We identify 48,415 enhancer regions and characterize their dynamics. We find that lineage commitment involves de novo establishment of 17,035 lineage-specific enhancers. These enhancer repertoire expansions foreshadow transcriptional programs in differentiated cells. Combining our enhancer catalog with gene expression profiles, we elucidate the transcription factor network controlling chromatin dynamics and lineage specification in hematopoiesis. Together, our results provide a comprehensive model of chromatin dynamics during development.
doi:10.1126/science.1256271
PMCID: PMC4412442  PMID: 25103404
4.  Massively parallel single cell RNA-Seq for marker-free decomposition of tissues into cell types 
Science (New York, N.Y.)  2014;343(6172):776-779.
In multi-cellular organisms, biological function emerges when heterogeneous cell types form complex organs. Nevertheless dissection of tissues into mixtures of cellular subpopulations is currently challenging. We introduce an automated massively parallel single-cell RNA sequencing approach for analyzing in vivo transcriptional states in thousands of single cells. Combined with unsupervised classification algorithms, this facilitates ab initio cell type characterization of splenic tissues. Modeling single-cell transcriptional states in dendritic cells and additional hematopoietic cell types uncovers rich cell-type heterogeneity and gene-modules activity in steady-state and after pathogen activation. Cellular diversity is thereby approached through inference of variable and dynamic pathway activity rather than a fixed pre-programmed cell-type hierarchy. These data demonstrate single-cell RNA-Seq as an effective tool for comprehensive cellular decomposition of complex tissues.
doi:10.1126/science.1247651
PMCID: PMC4412462  PMID: 24531970
5.  Progressive replacement of embryo-derived cardiac macrophages with age 
The Journal of Experimental Medicine  2014;211(11):2151-2158.
Molawi et al. examine the origin and cellular dynamics of macrophages in the heart during postnatal development. Cardiac macrophages derived from CX3CR1+ embryonic progenitors persist into adulthood, but the contribution of these cells to resident macrophages declines after birth with diminished self-renewal as the mice age. Over time, the heart is progressively reconstituted with bone marrow–derived macrophages, even in the absence of inflammation.
Cardiac macrophages (cMΦ) are critical for early postnatal heart regeneration and fibrotic repair in the adult heart, but their origins and cellular dynamics during postnatal development have not been well characterized. Tissue macrophages can be derived from embryonic progenitors or from monocytes during inflammation. We report that within the first weeks after birth, the embryo-derived population of resident CX3CR1+ cMΦ diversifies into MHCII+ and MHCII− cells. Genetic fate mapping demonstrated that cMΦ derived from CX3CR1+ embryonic progenitors persisted into adulthood but the initially high contribution to resident cMΦ declined after birth. Consistent with this, the early significant proliferation rate of resident cMΦ decreased with age upon diversification into subpopulations. Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative replacement of all cMΦ populations. Furthermore, parabiotic mice and BM chimeras of nonirradiated recipient mice revealed a slow but significant donor contribution to cMΦ. Together, our observations indicate that in the heart, embryo-derived cMΦ show declining self-renewal with age and are progressively substituted by monocyte-derived macrophages, even in the absence of inflammation.
doi:10.1084/jem.20140639
PMCID: PMC4203946  PMID: 25245760
6.  IL-23-mediated mononuclear phagocyte crosstalk protects mice from Citrobacter rodentium-induced colon immunopathology 
Nature Communications  2015;6:6525.
Gut homeostasis and mucosal immune defense rely on the differential contributions of dendritic cells (DC) and macrophages. Here we show that colonic CX3CR1+ mononuclear phagocytes are critical inducers of the innate response to Citrobacter rodentium infection. Specifically, the absence of IL-23 expression in macrophages or CD11b+ DC results in the impairment of IL-22 production and in acute lethality. Highlighting immunopathology as a death cause, infected animals are rescued by the neutralization of IL-12 or IFNγ. Moreover, mice are also protected when the CD103+ CD11b− DC compartment is rendered deficient for IL-12 production. We show that IL-12 production by colonic CD103+ CD11b− DC is repressed by IL-23. Collectively, in addition to its role in inducing IL-22 production, macrophage-derived or CD103− CD11b+ DC-derived IL-23 is required to negatively control the otherwise deleterious production of IL-12 by CD103+ CD11b− DC. Impairment of this critical mononuclear phagocyte crosstalk results in the generation of IFNγ-producing former TH17 cells and fatal immunopathology.
Macrophages and dendritic cells contribute to gut homeostasis and mucosal immune defense. Here, Aychek et al. describe an IL-23-based crosstalk between these cells that prevents lethal immunopathology during Citrobacter rodentium infection.
doi:10.1038/ncomms7525
PMCID: PMC4382688  PMID: 25761673
7.  Opposing Effects of Membrane-Anchored CX3CL1 on Amyloid and Tau Pathologies via the p38 MAPK Pathway 
The Journal of Neuroscience  2014;34(37):12538-12546.
Several Alzheimer's disease (AD) risk genes are specifically expressed by microglia within the CNS. However, the mechanisms by which microglia regulate the pathological hallmarks of AD—extracellular deposition of β-amyloid (Aβ) and intraneuronal hyperphosphorylation of microtubule-associated protein tau (MAPT)—remain to be established. Notably, deficiency for the microglial CX3CR1 receptor has opposing effects on Aβ and MAPT pathologies. CX3CL1, the neuronally derived cognate ligand for CX3CR1, signals both in membrane-anchored and soluble forms. In this study, we sought to determine the relative contribution on membrane-anchored versus soluble CX3CL1 in regulating the microglia-mediated amelioration of Aβ pathology, as well as provide insight into the potential downstream microglial-based mechanisms. As expected, CX3CL1 deficiency reduced Aβ deposition in APPPS1 animals in a similar manner to CX3CR1 deficiency. Surprisingly, however, CX3CL1-deficient APPPS1 animals exhibited enhanced neuronal MAPT phosphorylation despite reduced amyloid burden. Importantly, neither of these phenotypes was altered by transgenic expression of the soluble CX3CL1 isoform, suggesting that it is the membrane-anchored version of CX3CL1 that regulates microglial phagocytosis of Aβ and neuronal MAPT phosphorylation. Analysis of transcript levels in purified microglia isolated from APPPS1 mice with the various CX3CL1/CX3CR1 genotypes revealed increased expression of inflammatory cytokines and phagocytic markers, which was associated with activation of p38 mitogen-activated protein kinase and Aβ internalization within microglia. Together, these studies challenge the “frustrated phagocytosis” concept and suggest that neuronal–microglial communication link the two central AD pathologies.
doi:10.1523/JNEUROSCI.0853-14.2014
PMCID: PMC4160782  PMID: 25209291
Alzheimer's disease; CX3CL1; microglia; phagocytosis
8.  Guardians of the Gut – Murine Intestinal Macrophages and Dendritic Cells 
Intestinal mononuclear phagocytes find themselves in a unique environment, most prominently characterized by its constant exposure to commensal microbiota and food antigens. This anatomic setting has resulted in a number of specializations of the intestinal mononuclear phagocyte compartment that collectively contribute the unique steady state immune landscape of the healthy gut, including homeostatic innate lymphoid cells, B, and T cell compartments. As in other organs, macrophages and dendritic cells (DCs) orchestrate in addition the immune defense against pathogens, both in lymph nodes and mucosa-associated lymphoid tissue. Here, we will discuss origins and functions of intestinal DCs and macrophages and their respective subsets, focusing largely on the mouse and cells residing in the lamina propria.
doi:10.3389/fimmu.2015.00254
PMCID: PMC4451680  PMID: 26082775
gut; dendritic cells; macrophages; homeostasis; inflammation; IBD
9.  Recruitment of Beneficial M2 Macrophages to Injured Spinal Cord Is Orchestrated by Remote Brain Choroid Plexus 
Immunity  2013;38(3):555-569.
SUMMARY
Monocyte-derived macrophages are essential for recovery after spinal cord injury, but their homing mechanism is poorly understood. Here, we show that although of common origin, the homing of proinflammatory (M1) and the “alternatively activated” anti-inflammatory (M2) macrophages to traumatized spinal cord (SC) was distinctly regulated, neither being through breached blood-brain barrier. The M1 macrophages (Ly6chiCX3CR1lo) derived from monocytes homed in a CCL2 chemokine-dependent manner through the adjacent SC leptomeninges. The resolving M2 macrophages (Ly6cloCX3CR1hi) derived from monocytes trafficked through a remote blood-cerebrospinal-fluid (CSF) barrier, the brain-ventricular choroid plexus (CP), via VCAM-1-VLA-4 adhesion molecules and epithelial CD73 enzyme for extravasation and epithelial transmigration. Blockage of these determinants, or mechanical CSF flow obstruction, inhibited M2 macrophage recruitment and impaired motor-function recovery. The CP, along with the CSF and the central canal, provided an anti-inflammatory supporting milieu, potentially priming the trafficking monocytes. Overall, our finding demonstrates that the route of monocyte entry to central nervous system provides an instructional environment to shape their function.
doi:10.1016/j.immuni.2013.02.012
PMCID: PMC4115271  PMID: 23477737
10.  Paired immunoglobulin-like receptor A is an intrinsic, self-limiting suppressor of IL-5-induced eosinophil development 
Nature immunology  2013;15(1):36-44.
Summary
Eosinophilia is a hallmark characteristic of TH2-associated diseases and is critically regulated by the central eosinophil growth factor interleukin 5 (IL-5). Here we demonstrate that IL-5 activity in eosinophils was regulated by paired immunoglobulin-like receptor (PIR)-A and PIR-B. Upon self-recognition of β2M molecules, PIR-B served as a permissive checkpoint for IL-5-induced eosinophil development by suppressing the pro-apoptotic activities of PIR-A, which were mediated by the Grb2-Erk-Bim pathway. PIR-B-deficient bone marrow (BM) eosinophils underwent compartmentalized apoptosis, resulting in decreased blood eosinophilia in naïve, IL-5- and aeroallergen-challenged mice. Subsequently, Pirb−/− mice displayed impaired aeroallergen-induced lung eosinophilia and induction of lung TH2 responses. Collectively, these data uncovers an intrinsic, self-limiting pathway regulating IL-5-induced eosinophil expansion, which has broad implications for eosinophil-associated diseases.
doi:10.1038/ni.2757
PMCID: PMC3869881  PMID: 24212998
11.  miR-142 orchestrates a network of actin cytoskeleton regulators during megakaryopoiesis 
eLife  2014;3:e01964.
Genome-encoded microRNAs (miRNAs) provide a posttranscriptional regulatory layer that controls the differentiation and function of various cellular systems, including hematopoietic cells. miR-142 is one of the most prevalently expressed miRNAs within the hematopoietic lineage. To address the in vivo functions of miR-142, we utilized a novel reporter and a loss-of-function mouse allele that we have recently generated. In this study, we show that miR-142 is broadly expressed in the adult hematopoietic system. Our data further reveal that miR-142 is critical for megakaryopoiesis. Genetic ablation of miR-142 caused impaired megakaryocyte maturation, inhibition of polyploidization, abnormal proplatelet formation, and thrombocytopenia. Finally, we characterized a network of miR-142-3p targets which collectively control actin filament homeostasis, thereby ensuring proper execution of actin-dependent proplatelet formation. Our study reveals a pivotal role for miR-142 activity in megakaryocyte maturation and function, and demonstrates a critical contribution of a single miRNA in orchestrating cytoskeletal dynamics and normal hemostasis.
DOI: http://dx.doi.org/10.7554/eLife.01964.001
eLife digest
DNA carries all the information needed for life. This includes the codes required for making proteins, as well as instructions on when, where, and how much of these proteins need to be produced. There are a number of ways by which cells control protein manufacturing, one of which is based on small RNAs called microRNAs. Before proteins are assembled, the DNA molecule is copied into a temporary replica dubbed messenger RNA. microRNAs are able to recognize specific messenger RNA molecules and block protein production.
microRNAs serve a very important regulatory role in our bodies and are involved in virtually all cellular processes, including the production of all classes of blood and immune cells. Platelets seal injuries and prevent excessive bleeding by creating a clot at the location of a wound. Platelets are produced in huge cellular factories called megakaryocytes, which need to have a flexible and dynamic internal skeleton or cytoskeleton to produce the platelets.
Chapnik et al. focus on one specific microRNA gene, which is vital for the production and the function of several classes of blood and immune cells. Chapnik et al. created a mouse model that does not produce one specific microRNA—miR-142—and found that mutant mice produced fewer platelets than normal mice. Although one possible explanation for this is that the mutant mice also had fewer megakaryocytes than normal, Chapnik et al. unexpectedly found that the number of megakaryocytes was in fact higher. However, these megakaryocytes do not reach functional maturity, which is required for platelet production. Many of the megakaryocytes made by the mutant mice were also smaller than normal and had an unusual cytoskeleton.
Using a genomic approach and molecular tools, Chapnik et al. show that miR-142 affects the production of several of the proteins responsible for the dynamic flexibility of the cytoskeleton in mature megakaryocytes. Therefore, a single microRNA can target multiple different proteins that coordinate the same pathway in the cells that are critical for clotting and hence for human health.
miR-142 has also been suggested to have important functions in blood stem cells and in blood cancer (leukemia). Therefore, the new mouse model could be used to investigate many other facets of the blood and immune system. Further research could also focus on whether the same cytoskeletal network is in charge of miR-142 activity in other blood cells, or if miR-142 silences different targets in different cells.
DOI: http://dx.doi.org/10.7554/eLife.01964.002
doi:10.7554/eLife.01964
PMCID: PMC4067751  PMID: 24859754
microRNA; miR-142; actin; cytoskeleton; megakaryocytes; megakaryopoiesis; mouse
12.  On-site education of VEGF-recruited monocytes improves their performance as angiogenic and arteriogenic accessory cells 
The Journal of Experimental Medicine  2013;210(12):2611-2625.
VEGF-driven neovascularization transiently recruits Ly6Chigh monocytes, which subsequently alter their phenotype and exert angiogenic function to enlarge small vessels.
Adult neovascularization relies on the recruitment of monocytes to the target organ or tumor and functioning therein as a paracrine accessory. The exact origins of the recruited monocytes and the mechanisms underlying their plasticity remain unclear. Using a VEGF-based transgenic system in which genetically tagged monocytes are conditionally summoned to the liver as part of a VEGF-initiated angiogenic program, we show that these recruited cells are derived from the abundant pool of circulating Ly6Chi monocytes. Remarkably, however, upon arrival at the VEGF-induced organ, but not the naive organ, monocytes undergo multiple phenotypic and functional changes, endowing them with enhanced proangiogenic capabilities and, importantly, with a markedly increased capacity to remodel existing small vessels into larger conduits. Notably, monocytes do not differentiate into long-lived macrophages, but rather appear as transient accessory cells. Results from transfers of presorted subpopulations and a novel tandem transfer strategy ruled out selective recruitment of a dedicated preexisting subpopulation or onsite selection, thereby reinforcing active reprogramming as the underlying mechanism for improved performance. Collectively, this study uncovered a novel function of VEGF, namely, on-site education of recruited “standard” monocytes to become angiogenic and arteriogenic professional cells, a finding that may also lend itself for a better design of angiogenic therapies.
doi:10.1084/jem.20120690
PMCID: PMC3832929  PMID: 24166715
13.  Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis 
Immunity  2012;38(1):79-91.
SUMMARY
Mononuclear phagocytes, including monocytes, macrophages and dendritic cells, contribute to tissue integrity, as well as innate and adaptive immune defense. Emerging evidence for labour division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organisation of the cellular network are not well-defined. Here we report a fate mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX3CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue resident macrophage populations, including liver Kupffer cells, lung alveolar, splenic and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that the short-lived Ly6C+ monocytes constitute obligatory steady state precursors of blood-resident Ly6C− cells and that the abundance of Ly6C+ blood monocytes dynamically controls the circulation life span of their progeny.
doi:10.1016/j.immuni.2012.12.001
PMCID: PMC3908543  PMID: 23273845
14.  Transcriptional Reprogramming of CD11b+Esamhi Dendritic Cell Identity and Function by Loss of Runx3 
PLoS ONE  2013;8(10):e77490.
Classical dendritic cells (cDC) are specialized antigen-presenting cells mediating immunity and tolerance. cDC cell-lineage decisions are largely controlled by transcriptional factor regulatory cascades. Using an in vivo cell-specific targeting of Runx3 at various stages of DC lineage development we show that Runx3 is required for cell-identity, homeostasis and function of splenic Esamhi DC. Ablation of Runx3 in DC progenitors led to a substantial decrease in splenic CD4+/CD11b+ DC. Combined chromatin immunoprecipitation sequencing and gene expression analysis of purified DC-subsets revealed that Runx3 is a key gene expression regulator that facilitates specification and homeostasis of CD11b+Esamhi DC. Mechanistically, loss of Runx3 alters Esamhi DC gene expression to a signature characteristic of WT Esamlow DC. This transcriptional reprogramming caused a cellular change that diminished phagocytosis and hampered Runx3-/- Esamhi DC capacity to prime CD4+ T cells, attesting to the significant role of Runx3 in specifying Esamhi DC identity and function.
doi:10.1371/journal.pone.0077490
PMCID: PMC3817345  PMID: 24204843
15.  In Vivo Depletion of CD11c+ Dendritic Cells Abrogates Priming of CD8+ T Cells by Exogenous Cell-Associated Antigens 
Immunity  2002;17(2):211-220.
Summary
Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections. Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo. We show that in vivo DC are required to cross-prime CTL precursors. Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens. DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii.
PMCID: PMC3689299  PMID: 12196292
16.  Microglia, seen from the CX3CR1 angle 
Microglial cells in brain and spinal cord are characterized by high expression of the chemokine receptor CX3CR1. Expression of the sole CX3CR1 ligand, the membrane-tethered and sheddable chemokine CX3CL1/fractalkine, is restricted in the brain parenchyma to selected neurons. Here we summarize our current understanding of the physiological role of CX3CR1 for microglia function and the CX3C axis in microglial/neuronal crosstalk in homeostasis and under challenge. Moreover, we will discuss the efforts of our laboratory and others to exploit CX3CR1 promoter activity for the visualization and genetic manipulation of microglia to probe their functional contributions in the central nerve system (CNS) context.
doi:10.3389/fncel.2013.00026
PMCID: PMC3600435  PMID: 23507975
microglia; neuropathology; Cre-loxP knock-in mice; CX3CR1; neuroimmunology
17.  Notch2 receptor signaling controls functional differentiation of dendritic cells in the spleen and intestine 
Immunity  2011;35(5):780-791.
Summary
Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b+ DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esamhi DC subset required lymphotoxin beta receptor signaling, proliferated in situ and facilitated CD4+ T cell priming. The Notch-independent Esamlo DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b+ CD103+ DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4+ T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b+ DC subsets in the spleen and intestine.
doi:10.1016/j.immuni.2011.08.013
PMCID: PMC3225703  PMID: 22018469
18.  The Chemokine Receptor CX3CR1 Mediates Homing of MHC class II–Positive Cells to the Normal Mouse Corneal Epithelium 
Purpose
Recent investigations have revealed that populations of macrophages and dendritic cells (DCs) are present in the stroma and epithelium of the cornea, although the precise phenotype and distribution are still controversial. CX3CR1, the sole receptor for the chemokine fractalkine, is expressed by these monocyte-derived cells. Transgenic CX3CR1GFP mice, in which either one (heterozygous) or both (homozygous) copies of the CX3CR1 gene were replaced by enhanced green fluorescent protein (eGFP), were used to characterize monocyte-derived cells in the mouse cornea and to determine whether the expression of this receptor influences the recruitment of these cells into the normal cornea.
Methods
Wholemount corneas were immunostained with anti-leukocyte antibodies to the phenotypic markers major histocompatibility complex (MHC) class II, CD169, CD68, CD11b, and CD45 and analyzed by epifluorescence and confocal microscopy. The density of intraepithelial MHC class II+ cells was quantified in wild-type, CX3CR1+/GFP heterozygous, CX3CR1GFP/GFP homozygous, and CX3CR1-knockout mice.
Results
There was a significant reduction in the number of MHC class II+ cells (putative DCs) in the corneal epithelium of CX3CR1-deficient mice (P < 0.009) compared with wild-type mice, and the few cells that were present did not possess classic dendriform morphology. No GFP+ MHC class II– cells were noted in the epithelium. Dual immunostaining of corneas in both heterozygous and homozygous (CX3CR1-deficient) mice revealed GFP+ cells with a more pleomorphic morphology throughout the entire corneal stroma that were CD11b+ CD169+, and had variable degrees of expression of CD68 and MHC class II. The immunophenotype and morphology of these intrastromal cells is strongly indicative of a macrophage phenotype.
Conclusions
This study has identified a role for CX3CR1 in the normal recruitment of MHC class II+ putative DCs into the corneal epithelium and establishes a model for investigating monocyte-derived cells and fractalkine/CX3CR1 interactions during corneal disease.
doi:10.1167/iovs.06-0746
PMCID: PMC3392181  PMID: 17389486
19.  Mouse Dendritic Cells Pulsed with Capsular Polysaccharide Induce Resistance to Lethal Pneumococcal Challenge: Roles of T Cells and B Cells 
PLoS ONE  2012;7(6):e39193.
Mice are exceedingly sensitive to intra-peritoneal (IP) challenge with some virulent pneumococci (LD50 = 1 bacterium). To investigate how peripheral contact with bacterial capsular polysaccharide (PS) antigen can induce resistance, we pulsed bone marrow dendritic cells (BMDC) of C57BL/6 mice with type 4 or type 3 PS, injected the BMDC intra-foot pad (IFP) and challenged the mice IP with supra-lethal doses of pneumococci. We examined the responses of T cells and B cells in the draining popliteal lymph node and measured the effects on the bacteria in the peritoneum and blood. We now report that: 1) The PS co-localized with MHC molecules on the BMDC surface; 2) PS-specific T and B cell proliferation and IFNγ secretion was detected in the draining popliteal lymph nodes on day 4; 3) Type-specific resistance to lethal IP challenge was manifested only after day 5; 4) Type-specific IgM and IgG antibodies were detected in the sera of only some of the mice, but B cells were essential for resistance; 5) Control mice vaccinated with a single injection of soluble PS did not develop a response in the draining popliteal lymph node and were not protected; 6) Mice injected with unpulsed BMDC also did not resist challenge: In unprotected mice, pneumococci entered the blood shortly after IP inoculation and multiplied exponentially in both blood and peritoneum killing the mice within 20 hours. Mice vaccinated with PS-pulsed BMDC trapped the bacteria in the peritoneum. The trapped bacteria proliferated exponentially IP, but died suddenly at 18–20 hours. Thus, a single injection of PS antigen associated with intact BMDC is a more effective vaccine than the soluble PS alone. This model system provides a platform for studying novel aspects of PS-targeted vaccination.
doi:10.1371/journal.pone.0039193
PMCID: PMC3377650  PMID: 22723962
20.  Dissecting the Autocrine and Paracrine Roles of the CCR2-CCL2 Axis in Tumor Survival and Angiogenesis 
PLoS ONE  2012;7(1):e28305.
The CCL2 CCR2 axis is likely to contributes to the development and progression of cancer diseases by two major mechanisms; autocrine effect of CCL2 as a survival/growth factor for CCR2+ cancer cells and, the attraction of CCR2+ CX3CR1+tumor associated macrophages that in the absence of CCR2 hardly migrate. Thus far no in vivo system has been set up to differentiate the selective contribution of each of these features to cancer development. Here we employed a chimera animal model in which all non-malignant cells are CCR2−/−, but all cancer cells are CCR2+, combined with an adoptive transfer system of bone marrow (BM) CX3CR1+ cells from CCR2+ mice harboring a targeted replacement of the CX3CR1gene by an enhanced green fluorescent protein (EGFP) reporter gene (cx3cr1gfp), together with the CD45.1 congene. Using this system we dissected the selective contribution of CX3CR1+CCR2+ cells, which comprise only about 7% of CD11b+ BM cells, to tumor development and angiogenesis. Showing that aside for their direct pro-angiogenic effect they are essential for the recruitment of other CD11b+ cells to the tumor site. We further show that the administration of CCR2-Ig, that selectively and specifically neutralize CCL2, to mice in which CCR2 is expressed only on tumor cells, further suppressed tumor development, implicating for the key role of this chemokine supporting tumor survival in an autocrine manner. This further emphasizes the important role of CCL2 as a target for therapy of cancer diseases.
doi:10.1371/journal.pone.0028305
PMCID: PMC3261135  PMID: 22279523
21.  Non-Identical Twins – Microglia and Monocyte-Derived Macrophages in Acute Injury and Autoimmune Inflammation 
The brain has been commonly regarded as a “tissue behind walls.” Appearance of immune cells in the brain has been taken as a sign of pathology. Moreover, since infiltrating monocyte-derived macrophages and activated resident microglia were indistinguishable by conventional means, both populations were considered together as inflammatory cells that should be mitigated. Yet, because the microglia permanently reside in the brain, attributing to them negative properties evoked an ongoing debate; why cells that are supposed to be the brain guardians acquire only destructive potential? Studies over the last two decades in the immune arena in general, and in the context of central nervous system pathology in particular, have resulted in a paradigm shift toward a more balanced appreciation of the contributions of immune cells in the context of brain maintenance and repair, and toward the recognition of distinct roles of resident microglia and infiltrating monocyte-derived macrophages.
doi:10.3389/fimmu.2012.00089
PMCID: PMC3345364  PMID: 22566968
microglia; monocyte-derived macrophages; central nervous system; neurodegeneration; neuroinflammation
22.  Utilization of Murine Colonoscopy for Orthotopic Implantation of Colorectal Cancer 
PLoS ONE  2011;6(12):e28858.
Background
Colorectal-cancer (CRC) research has greatly benefited from the availability of small animal tumor models. Spontaneous and chemically-induced CRC models are widely used yet limited in their resemblance to human disease and are often prolonged, not accurately repetitive, and associated with inflammatory side effects. In-situ murine or human tumor implantation in the gastrointestinal tract of mice is extremely challenging, and limited by inter-animal variability and procedure-related complications and mortality. As a result, in frequent studies CRC is implanted in distal sites, most commonly the subcutaneous region, an approach that is greatly limited by the absence of normal gastrointestinal tumor milieu and has substantial effects on tumor development.
Aims
In this study we aimed to develop a well-tolerated repetitive tool to study CRC in small animals by adapting the murine colonoscopy system to serve as a platform for colonic sub-mucosal orthotopic implantation of human and murine CRC tumor cells.
Results
We report the establishment of a novel small-animal CRC model that is minimally invasive, rapid, well-tolerated, highly reproducible, and confers precise control of tumor number, location and growth rate. Moreover, we show that this model uniquely allows the side-by-side induction of distinct genetically manipulated tumors, enabling the mechanistic study of tumor interaction and cross-talk within the native intestinal microenvironment.
Conclusions
Employment of this new approach may represent a major technical advance for the in-vivo study of CRC.
doi:10.1371/journal.pone.0028858
PMCID: PMC3236220  PMID: 22174916
23.  Coupled pre-mRNA and mRNA dynamics unveil operational strategies underlying transcriptional responses to stimuli 
Genome-wide simultaneous measurements of pre-mRNA and mRNA expression reveal unexpected time-dependent transcript production and degradation profiles in response to external stimulus, as well as a striking lack of concordance between mRNA abundance and transcript production profiles.
By analyzing the signals from intronic probes of exon arrays, we performed, for the first time, genome-wide measurement of pre-mRNA expression dynamics.We discovered a striking lack of correspondence between mRNA and pre-mRNA temporal expression profiles following stimulus, demonstrating that measurement of mRNA dynamics does not suffice to infer transcript production profiles.By combining simultaneous measurement of pre-mRNA and mRNA profiles with a simple new quantitative theoretical description of transcription, we are able to infer complex time dependence of both transcript production and mRNA degradation.The production profiles of many transcripts reveal an operational strategy we termed Production Overshoot, which is used to accelerate mRNA response. The biological relevance of our findings was substantiated by observing similar results when studying the response of three different mammalian cell types to different stimuli.
Transcriptional responses to extracellular stimuli involve tuning the rates of transcript production and degradation. Here, we show that the time-dependent profiles of these rates can be inferred from simultaneous measurements of precursor mRNA (pre-mRNA) and mature mRNA profiles. Transcriptome-wide measurements demonstrate that genes with similar mRNA profiles often exhibit marked differences in the amplitude and onset of their production rate. The latter is characterized by a large dynamic range, with a group of genes exhibiting an unexpectedly strong transient production overshoot, thereby accelerating their induction and, when combined with time-dependent degradation, shaping transient responses with precise timing and amplitude.
doi:10.1038/msb.2011.62
PMCID: PMC3202801  PMID: 21915116
half-life; operational strategy; pre-mRNA; transcriptional response
24.  The Natural Cytotoxicity Receptor 1 Contribution to Early Clearance of Streptococcus pneumoniae and to Natural Killer-Macrophage Cross Talk 
PLoS ONE  2011;6(8):e23472.
Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligandhigh lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-liganddull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC.
doi:10.1371/journal.pone.0023472
PMCID: PMC3161738  PMID: 21887255
25.  Neuroprotection and progenitor cell renewal in the injured adult murine retina requires healing monocyte-derived macrophages 
After retinal injury in mice, infiltrating monocyte-derived macrophages preserve retinal ganglion cells and promote retinal progenitor cell renewal.
The death of retinal ganglion cells (RGCs) is a hallmark of many retinal neuropathies. Neuroprotection, axonal regeneration, and cell renewal are vital for the integrity of the visual system after insult but are scarce in the adult mammalian retina. We hypothesized that monocyte-derived macrophages, known to promote healing in peripheral tissues, are required after an insult to the visual system, where their role has been largely overlooked. We found that after glutamate eye intoxication, monocyte-derived macrophages infiltrated the damaged retina of mice. Inhibition of this infiltration resulted in reduced survival of RGCs and diminished numbers of proliferating retinal progenitor cells (RPCs) in the ciliary body. Enhancement of the circulating monocyte pool led to increased RGC survival and RPC renewal. The infiltrating monocyte-derived macrophages skewed the milieu of the injured retina toward an antiinflammatory and neuroprotective one and down-regulated accumulation of other immune cells, thereby resolving local inflammation. The beneficial effect on RGC survival depended on expression of interleukin 10 and major histocompatibility complex class II molecules by monocyte-derived macrophages. Thus, we attribute to infiltrating monocyte-derived macrophages a novel role in neuroprotection and progenitor cell renewal in the injured retina, with far-reaching potential implications to retinal neuropathies and other neurodegenerative disorders.
doi:10.1084/jem.20101202
PMCID: PMC3023128  PMID: 21220455

Results 1-25 (46)