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1.  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
2.  Paired immunoglobulin-like receptor A is an intrinsic, self-limiting suppressor of IL-5-induced eosinophil development 
Nature immunology  2013;15(1):10.1038/ni.2757.
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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  CCL17-expressing dendritic cells drive atherosclerosis by restraining regulatory T cell homeostasis in mice 
The Journal of Clinical Investigation  2011;121(7):2898-2910.
Immune mechanisms are known to control the pathogenesis of atherosclerosis. However, the exact role of DCs, which are essential for priming of immune responses, remains elusive. We have shown here that the DC-derived chemokine CCL17 is present in advanced human and mouse atherosclerosis and that CCL17+ DCs accumulate in atherosclerotic lesions. In atherosclerosis-prone mice, Ccl17 deficiency entailed a reduction of atherosclerosis, which was dependent on Tregs. Expression of CCL17 by DCs limited the expansion of Tregs by restricting their maintenance and precipitated atherosclerosis in a mechanism conferred by T cells. Conversely, a blocking antibody specific for CCL17 expanded Tregs and reduced atheroprogression. Our data identify DC-derived CCL17 as a central regulator of Treg homeostasis, implicate DCs and their effector functions in atherogenesis, and suggest that CCL17 might be a target for vascular therapy.
doi:10.1172/JCI44925
PMCID: PMC3223829  PMID: 21633167
19.  Development of monocytes, macrophages and dendritic cells 
Science (New York, N.Y.)  2010;327(5966):656-661.
Monocytes and macrophages are critical effectors and regulators of inflammation and the innate immune response, the immediate, pre-programmed arm of the immune system. Dendritic cells initiate and regulate the highly pathogen-specific adaptive immune responses, and are central to the development of immunologic memory and tolerance. Recent in vivo experimental approaches in the mouse have unveiled new aspects of the developmental and lineage relationships among these cell populations. Despite this, the origin and differentiation cues for many tissue macrophages, monocytes, and dendritic cell subsets in mice, and the corresponding cell populations in humans, remain to be elucidated.
doi:10.1126/science.1178331
PMCID: PMC2887389  PMID: 20133564
20.  Therapy of Murine Pulmonary Aspergillosis with Antibody-Alliinase Conjugates and Alliin▿  
Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The high morbidity and mortality rates as well as the poor efficacy of antifungal agents remain major clinical concerns. Allicin (diallyl-dithiosulfinate), which is produced by the garlic enzyme alliinase from the harmless substrate alliin, has been shown to have wide-range antifungal specificity. A monoclonal antibody (MAb) against A. fumigatus was produced and chemically ligated to the enzyme alliinase. The purified antibody-alliinase conjugate bound to conidia and hyphae of A. fumigatus at nanomolar concentrations. In the presence of alliin, the conjugate produced cytotoxic allicin molecules, which killed the fungus. In vivo testing of the therapeutical potential of the conjugate was carried out in immunosuppressed mice infected intranasally with conidia of A. fumigatus. Intratracheal (i.t.) instillation of the conjugate and alliin (four treatments) resulted in 80 to 85% animal survival (36 days), with almost complete fungal clearance. Repetitive intratracheal administration of the conjugate and alliin was also effective when treatments were initiated at a more advanced stage of infection (50 h). The fungi were killed specifically without causing damage to the lung tissue or overt discomfort to the animals. Intratracheal instillation of the conjugate without alliin or of the unconjugated monoclonal antibody significantly delayed the death of the infected mice, but only 20% of the animals survived. A limitation of this study is that the demonstration was achieved in a constrained setting. Other routes of drug delivery will be investigated for the treatment of pulmonary and extrapulmonary aspergillosis.
doi:10.1128/AAC.01267-09
PMCID: PMC2812126  PMID: 19949059
21.  Infiltrating Blood-Derived Macrophages Are Vital Cells Playing an Anti-inflammatory Role in Recovery from Spinal Cord Injury in Mice 
PLoS Medicine  2009;6(7):e1000113.
Using a mouse model of spinal injury, Michal Schwartz and colleagues tested the effect of macrophages on the recovery process and demonstrate an important anti-inflammatory role for a subset of infiltrating monocyte-derived macrophages that is dependent upon their expression of interleukin 10.
Background
Although macrophages (MΦ) are known as essential players in wound healing, their contribution to recovery from spinal cord injury (SCI) is a subject of debate. The difficulties in distinguishing between different MΦ subpopulations at the lesion site have further contributed to the controversy and led to the common view of MΦ as functionally homogenous. Given the massive accumulation in the injured spinal cord of activated resident microglia, which are the native immune occupants of the central nervous system (CNS), the recruitment of additional infiltrating monocytes from the peripheral blood seems puzzling. A key question that remains is whether the infiltrating monocyte-derived MΦ contribute to repair, or represent an unavoidable detrimental response. The hypothesis of the current study is that a specific population of infiltrating monocyte-derived MΦ is functionally distinct from the inflammatory resident microglia and is essential for recovery from SCI.
Methods and Findings
We inflicted SCI in adult mice, and tested the effect of infiltrating monocyte-derived MΦ on the recovery process. Adoptive transfer experiments and bone marrow chimeras were used to functionally distinguish between the resident microglia and the infiltrating monocyte-derived MΦ. We followed the infiltration of the monocyte-derived MΦ to the injured site and characterized their spatial distribution and phenotype. Increasing the naïve monocyte pool by either adoptive transfer or CNS-specific vaccination resulted in a higher number of spontaneously recruited cells and improved recovery. Selective ablation of infiltrating monocyte-derived MΦ following SCI while sparing the resident microglia, using either antibody-mediated depletion or conditional ablation by diphtheria toxin, impaired recovery. Reconstitution of the peripheral blood with monocytes resistant to ablation restored the lost motor functions. Importantly, the infiltrating monocyte-derived MΦ displayed a local anti-inflammatory beneficial role, which was critically dependent upon their expression of interleukin 10.
Conclusions
The results of this study attribute a novel anti-inflammatory role to a unique subset of infiltrating monocyte-derived MΦ in SCI recovery, which cannot be provided by the activated resident microglia. According to our results, limited recovery following SCI can be attributed in part to the inadequate, untimely, spontaneous recruitment of monocytes. This process is amenable to boosting either by active vaccination with a myelin-derived altered peptide ligand, which indicates involvement of adaptive immunity in monocyte recruitment, or by augmenting the naïve monocyte pool in the peripheral blood. Thus, our study sheds new light on the long-held debate regarding the contribution of MΦ to recovery from CNS injuries, and has potentially far-reaching therapeutic implications.
Please see later in the article for Editors' Summary
Editors' Summary
Background
Every year, spinal cord injuries paralyze about 11,000 people in the US. The spinal cord, which contains bundles of nervous system cells called neurons, is the communication highway between the brain and the body. Messages from the brain travel down the spinal cord to control movement, breathing and other bodily functions; messages from the skin and other sensory organs travel up the spinal cord to keep the brain informed about the body. The bones of the spine normally protect the spinal cord but, if these are broken or displaced, the spinal cord can be cut or compressed, which interrupts the information flow. Damage near the top of the spinal cord paralyzes the arms and legs (tetraplegia); damage lower down paralyzes the legs only (paraplegia). Spinal cord injuries also cause other medical problems, including the loss of bladder and bowel control. Currently, there is no effective treatment for spinal cord injuries, which usually cause permanent disability because the damaged nerve fibers rarely regrow.
Why Was This Study Done?
After a spinal cord injury, immune system cells called macrophages accumulate at the injury site. Some of these macrophages—so-called monocyte-derived macrophages—come into (infiltrate) the spinal cord from the blood in response to the injury, whereas others—microglia—are always in the nervous system. Although macrophages are essential for wound healing in other parts of the body, it is unclear whether they have good or bad effects in the spinal cord. Many experts believe that immune system cells hinder healing in the spinal cord and should be suppressed or eliminated, but other scientists claim that macrophages secrete factors that stimulate nerve regrowth. Furthermore, although some macrophages elsewhere in the body have proinflammatory (potentially deleterious) effects, others have anti-inflammatory (beneficial) effects. So do the infiltrating monocyte-derived macrophages and the resident microglia (which are proinflammatory) have different functions at spinal cord injury sites? In this study, the researchers try to answer this important question.
What Did the Researchers Do and Find?
The researchers bruised a small section of the spinal cord of adult mice and then investigated the effect of infiltrating monocyte-derived macrophages on the recovery process. Monocyte-derived macrophages and microglia cannot be distinguished using standard staining techniques so to study their behavior after spinal cord injury the researchers introduced labeled monocyte-derived macrophages into their experimental animals by using adoptive transfer (injection of genetically labeled monocytes into the animals) or by making bone marrow chimeras. In this second technique, the animals' monocyte-derived macrophages (but not their microglia) were killed by irradiating the animals before injection of genetically labeled bone marrow, the source of monocytes. Using these approaches, the researchers found that monocyte-derived macrophages collected at the margins of spinal cord injury sites whereas microglia accumulated throughout the sites. When the pool of monocyte-derived macrophages in the mice was increased by adoptive transfer or by using a technique called “CNS-specific vaccination,” more monocyte-derived macrophages infiltrated the injury site and the animals' physical recovery from injury improved. Conversely, removal of the infiltrating monocyte-derived macrophages from the injury site reduced the animals' physical recovery. Other experiments indicated that the infiltrating monocyte-derived macrophages have a beneficial, local anti-inflammatory effect that is dependent on their expression of interleukin-10 (an anti-inflammatory signaling molecule).
What Do These Findings Mean?
These findings provide new information about the contribution of monocyte-derived macrophages to spontaneous recovery from spinal cord injury, a contribution that has long been debated. In particular, the findings suggest that this subset of macrophages (but not the resident microglia) has a beneficial effect on spinal cord injuries that is mediated by their production of the anti-inflammatory molecule interleukin-10. The findings also show that the effect of these monocyte-derived macrophages can be boosted, at least in mice. Although results obtained in experiments done in animals do not always accurately reflect what happens in people, this new understanding of the different functions of microglia and infiltrating monocyte-derived macrophages after injury to the spinal cord may eventually lead to the development of better treatments for spinal cord injuries.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000113.
The MedlinePlus encyclopedia provides information about spinal cord injuries (in English and Spanish)
The US National Institute of Neurological Disorders and Stroke provides detailed information about spinal cord injury, including information on current research into the problem (in English and Spanish)
MedlinePlus provides an interactive tutorial on spinal cord injury and a list of links to additional information (in English and Spanish)
doi:10.1371/journal.pmed.1000113
PMCID: PMC2707628  PMID: 19636355
22.  Efficient Clearance of Aspergillus fumigatus in Murine Lungs by an Ultrashort Antimicrobial Lipopeptide, Palmitoyl-Lys-Ala-dAla-Lys ▿  
Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The inefficiency of antifungal agents and high mortality rate resulting from invasive aspergillosis remain major clinical concerns. Recently, we reported on a new family of ultrashort cationic lipopeptides active in vitro against fungi. Mode of action studies supported a membranolytic or a detergent-like effect. Here, we screened several lipopeptides in vitro for their anti-A. fumigatus activity. To investigate the therapeutic properties of the selected peptides in vivo, we challenged immunosuppressed C57BL/6 wild-type mice intranasally with DsRed-labeled A. fumigatus conidia and subsequently treated the animals locally with the lipopeptides. Confocal microscopic analysis revealed the degradation of DsRed-labeled hyphal forms and residual conidia in the lungs of the mice. The most efficient peptide was tested further using a survival assay and was found to significantly prolong the life of the treated animals, whereas no mice survived with the current standard antifungal treatment with amphotericin B. Moreover, as opposed to the drug-treated lungs, the peptide-treated lungs did not display any toxicity of the peptide. Our results highlight the potential of this family of lipopeptides for the treatment of pulmonary invasive aspergillosis.
doi:10.1128/AAC.00526-08
PMCID: PMC2533451  PMID: 18606837
23.  Uterine DCs are crucial for decidua formation during embryo implantation in mice 
The Journal of Clinical Investigation  2008;118(12):3954-3965.
Implantation is a key stage during pregnancy, as the fate of the embryo is often decided upon its first contact with the maternal endometrium. Around this time, DCs accumulate in the uterus; however, their role in pregnancy and, more specifically, implantation, remains unknown. We investigated the function of uterine DCs (uDCs) during implantation using a transgenic mouse model that allows conditional ablation of uDCs in a spatially and temporally regulated manner. Depletion of uDCs resulted in a severe impairment of the implantation process, leading to embryo resorption. Depletion of uDCs also caused embryo resorption in syngeneic and T cell–deficient pregnancies, which argues against a failure to establish immunological tolerance during implantation. Moreover, even in the absence of embryos, experimentally induced deciduae failed to adequately form. Implantation failure was associated with impaired decidual proliferation and differentiation. Dynamic contrast-enhanced MRI revealed perturbed angiogenesis characterized by reduced vascular expansion and attenuated maturation. We suggest therefore that uDCs directly fine-tune decidual angiogenesis by providing two critical factors, sFlt1 and TGF-β1, that promote coordinated blood vessel maturation. Collectively, uDCs appear to govern uterine receptivity, independent of their predicted role in immunological tolerance, by regulating tissue remodeling and angiogenesis. Importantly, our results may aid in understanding the limited implantation success of embryos transferred following in vitro fertilization.
doi:10.1172/JCI36682
PMCID: PMC2582932  PMID: 19033665
24.  Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells 
Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)–resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c+ monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting “nature's adjuvant,” the inflammatory DC through induction of the endogenous danger signal uric acid.
doi:10.1084/jem.20071087
PMCID: PMC2292225  PMID: 18362170
25.  Microbe sampling by mucosal dendritic cells is a discrete, MyD88-independent stepin ΔinvG S. Typhimurium colitis 
Intestinal dendritic cells (DCs) are believed to sample and present commensal bacteria to the gut-associated immune system to maintain immune homeostasis. How antigen sampling pathways handle intestinal pathogens remains elusive. We present a murine colitogenic Salmonella infection model that is highly dependent on DCs. Conditional DC depletion experiments revealed that intestinal virulence of S. Typhimurium SL1344 ΔinvG mutant lacking a functional type 3 secretion system-1 (ΔinvG)critically required DCs for invasion across the epithelium. The DC-dependency was limited to the early phase of infection when bacteria colocalized with CD11c+CX3CR1+ mucosal DCs. At later stages, the bacteria became associated with other (CD11c−CX3CR1−) lamina propria cells, DC depletion no longer attenuated the pathology, and a MyD88-dependent mucosal inflammation was initiated. Using bone marrow chimeric mice, we showed that the MyD88 signaling within hematopoietic cells, which are distinct from DCs, was required and sufficient for induction of the colitis. Moreover, MyD88-deficient DCs supported transepithelial uptake of the bacteria and the induction of MyD88-dependent colitis. These results establish that pathogen sampling by DCs is a discrete, and MyD88-independent, step during the initiation of a mucosal innate immune response to bacterial infection in vivo.
doi:10.1084/jem.20070633
PMCID: PMC2271026  PMID: 18268033

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