Classical monocyte ontogeny dictates their functions and fates as tissue macrophages.

Classical monocytes (CMs) are ephemeral myeloid immune cells that circulate in the blood. Emerging evidence suggests that CMs can have distinct ontogeny and originate from either granulocyte-monocyte- or monocyte-dendritic-cell progenitors (GMPs or MDPs). Here, we report surface markers that allowed segregation of murine GMP- and MDP-derived CMs, i.e., GMP-Mo and MDP-Mo, as well as their functional characterization, including fate definition following adoptive cell transfer. GMP-Mo and MDP-Mo yielded an equal increase in homeostatic CM progeny, such as blood-resident non-classical monocytes and gut macrophages; however, these cells differentially seeded various other selected tissues, including the dura mater and lung. Specifically, GMP-Mo and MDP-Mo differentiated into distinct interstitial lung macrophages, linking CM dichotomy to previously reported pulmonary macrophage heterogeneity. Collectively, we provide evidence for the existence of two functionally distinct CM subsets in the mouse that differentially contribute to peripheral tissue macrophage populations in homeostasis and following challenge.

Translatome Profiling of Tissue-Resident Macrophages Using the RiboTag Approach.

Global gene expression profiling has provided valuable insights into the specific contributions of different cell types to various physiological processes. Notably though, both bulk and single-cell transcriptomics require the prior retrieval of the cells from their tissue context to be analyzed. Isolation protocols for tissue macrophages are, however, notoriously inefficient and, moreover, prone to introduce considerable bias and artifacts. Here, we will discuss a valuable alternative, originally introduced by Amieux and colleagues. This so-called RiboTag approach allows, in combination with respective macrophage-specific Cre transgenic lines, to retrieve macrophage translatomes from crude tissue extracts. We will review our experience with this ingenious method, focusing on the study of brain macrophages, including microglia and border-associated cells. We will elaborate on the advantages of the RiboTag approach that render it a valuable complement to standard cell sorting-based profiling strategies, especially for the investigation of tissue macrophages.

Tackling Tissue Macrophage Heterogeneity by SplitCre Transgenesis.

Macrophages represent a broad spectrum of distinct, but closely related tissue-resident immune cells. This presents a major challenge for the study of functional aspects of these cells using classical Cre recombinase-mediated conditional mutagenesis in mice, since single promoter-driven Cre transgenic models often display limited specificity toward their intended target. The advent of CRISPR/Cas9 technology has now provided a time- and cost-effective method to explore the full potential of binary transgenic, intersectional genetics. Specifically, the use of two promoters driving inactive Cre fragments that, when co-expressed, dimerize and only then gain recombinase activity allows the characterization and manipulation of genetically defined tissue macrophage subpopulations. Here, we will elaborate on the use of this protocol to capitalize on these recent technological advances in mouse genetics and discuss their strengths and pitfalls to improve the study of tissue macrophage subpopulations in physiology and pathophysiology.

Perivascular cells induce microglial phagocytic states and synaptic engulfment via SPP1 in mouse models of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-ß oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.

Cognate microglia-T cell interactions shape the functional regulatory T cell pool in experimental autoimmune encephalomyelitis pathology.

Microglia, the parenchymal brain macrophages of the central nervous system, have emerged as critical players in brain development and homeostasis. The immune functions of these cells, however, remain less well defined. We investigated contributions of microglia in a relapsing-remitting multiple sclerosis paradigm, experimental autoimmune encephalitis in C57BL/6 x SJL F1 mice. Fate mapping-assisted translatome profiling during the relapsing-remitting disease course revealed the potential of microglia to interact with T cells through antigen presentation, costimulation and coinhibition. Abundant microglia-T cell aggregates, as observed by histology and flow cytometry, supported the idea of functional interactions of microglia and T cells during remission, with a bias towards regulatory T cells. Finally, microglia-restricted interferon-γ receptor and major histocompatibility complex mutagenesis significantly affected the functionality of the regulatory T cell compartment in the diseased central nervous system and remission. Collectively, our data establish critical non-redundant cognate and cytokine-mediated interactions of microglia with CD4+ T cells during autoimmune neuroinflammation.

Enteric glial cells favor accumulation of anti-inflammatory macrophages during the resolution of muscularis inflammation.

Monocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which micro-environmental factors are responsible for monocyte recruitment and anti-inflammatory Mφ differentiation in this paradigm. Here, we investigate Mφ heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue-protective Mφs. Results showed that muscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c+ monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving Mφ subpopulations during the resolution of muscularis inflammation, i.e. Cd206+ MhcIIhi and Timp2+ MhcIIlo Mφs. Interestingly, we found that damage to the micro-environment upon muscularis inflammation resulted in EGC activation, which in turn stimulated monocyte infiltration and the consequent differentiation in anti-inflammatory CD206+ Mφs via CCL2 and CSF1, respectively. In addition, CSF1-CSF1R signaling was shown to be essential for the differentiation of monocytes into CD206+ Mφs and EGC proliferation during muscularis inflammation. Our study provides a comprehensive insight into pro-resolving Mφ differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mφs, thereby highlighting pro-resolving Mφ differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.

A Binary Cre Transgenic Approach Dissects Microglia and CNS Border-Associated Macrophages.

The developmental and molecular heterogeneity of tissue macrophages is unravelling, as are their diverse contributions to physiology and pathophysiology. Moreover, also given tissues harbor macrophages in discrete anatomic locations. Functional contributions of specific cell populations can in mice be dissected using Cre recombinase-mediated mutagenesis. However, single promoter-based Cre models show limited specificity for cell types. Focusing on macrophages in the brain, we establish here a binary transgenic system involving complementation-competent NCre and CCre fragments whose expression is driven by distinct promoters: Sall1ncre: Cx3cr1ccre mice specifically target parenchymal microglia and compound transgenic Lyve1ncre: Cx3cr1ccre animals target vasculature-associated macrophages, in the brain, as well as other tissues. We imaged the respective cell populations and retrieved their specific translatomes using the RiboTag in order to define them and analyze their differential responses to a challenge. Collectively, we establish the value of binary transgenesis to dissect tissue macrophage compartments and their functions.

Interleukin-10 Prevents Pathological Microglia Hyperactivation following Peripheral Endotoxin Challenge.

Microglia, the resident macrophages of the brain parenchyma, are key players in central nervous system (CNS) development, homeostasis, and disorders. Distinct brain pathologies seem associated with discrete microglia activation modules. How microglia regain quiescence following challenges remains less understood. Here, we explored the role of the interleukin-10 (IL-10) axis in restoring murine microglia homeostasis following a peripheral endotoxin challenge. Specifically, we show that lipopolysaccharide (LPS)-challenged mice harboring IL-10 receptor-deficient microglia displayed neuronal impairment and succumbed to fatal sickness. Addition of a microglial tumor necrosis factor (TNF) deficiency rescued these animals, suggesting a microglia-based circuit driving pathology. Single cell transcriptome analysis revealed various IL-10 producing immune cells in the CNS, including most prominently Ly49D+ NK cells and neutrophils, but not microglia. Collectively, we define kinetics of the microglia response to peripheral endotoxin challenge, including their activation and robust silencing, and highlight the critical role of non-microglial IL-10 in preventing deleterious microglia hyperactivation.

Graft-versus-host disease of the CNS is mediated by TNF upregulation in microglia.

Acute graft-versus-host disease (GVHD) can affect the central nervous system (CNS). The role of microglia in CNS-GVHD remains undefined. In agreement with microglia activation, we found that profound morphological changes and MHC-II and CD80 upregulation occurred upon GVHD induction. RNA sequencing-based analysis of purified microglia obtained from mice with CNS-GVHD revealed TNF upregulation. Selective TNF gene deletion in microglia of Cx3cr1creER Tnffl/- mice reduced MHC-II expression and decreased CNS T cell infiltrates and VCAM-1+ endothelial cells. GVHD increased microglia TGF-ß-activated kinase-1 (TAK1) activation and NF-κB/p38 MAPK signaling. Selective Tak1 deletion in microglia using Cx3cr1creER Tak1fl/fl mice resulted in reduced TNF production and microglial MHC-II and improved neurocognitive activity. Pharmacological TAK1 inhibition reduced TNF production and MHC-II expression by microglia, Th1 and Th17 T cell infiltrates, and VCAM-1+ endothelial cells and improved neurocognitive activity, without blocking graft-versus-leukemia effects. Consistent with these findings in mice, we observed increased activation and TNF production of microglia in the CNS of GVHD patients. In summary, we prove a role for microglia in CNS-GVHD, identify the TAK1/TNF/MHC-II axis as a mediator of CNS-GVHD, and provide a TAK1 inhibitor-based approach against GVHD-induced neurotoxicity.

Comparative analysis of CreER transgenic mice for the study of brain macrophages: A case study.

Conditional mutagenesis and fate mapping have contributed considerably to our understanding of physiology and pathology. Specifically, Cre recombinase-based approaches allow the definition of cell type-specific contributions to disease development and of inter-cellular communication circuits in respective animal models. Here we compared Cx3 cr1CreER and Sall1CreER transgenic mice and their use to decipher the brain macrophage compartment as a showcase to discuss recent technological advances. Specifically, we highlight the need to define the accuracy of Cre recombinase expression, as well as strengths and pitfalls of these particular systems that should be taken into consideration when applying these models.

An Energy-Based Unified Approach to Predict the Low-Cycle Fatigue Life of Type 316L Stainless Steel under Various Temperatures and Strain-Rates.

An energy-based low-cycle fatigue model was proposed for applications at a range of temperatures. An existing model was extended to the integrated approach, incorporating the simultaneous effects of strain rate and temperature. A favored material at high temperature, type 316L stainless steel, was selected in this study and its material characteristics were investigated. Tensile tests and low-cycle fatigue tests were performed using several strain rates at a temperature ranging from room temperature to 650 °C. Material properties were obtained in terms of temperature using the displacement-controlled tensile tests and further material response were investigated using strain-controlled tensile tests. Consequently, no pronounced reduction in strengths occurred at temperatures between 300 and 550 °C, and a negative strain rate response was observed in the temperature range. Based on the low-cycle fatigue tests by varying strain rates and temperature, it was found that a normalized plastic strain energy density and a strain-rate modified cycle were successfully correlated. The accuracy of the model was discussed by comparing between predicted and experimental lives.

Engrafted parenchymal brain macrophages differ from microglia in transcriptome, chromatin landscape and response to challenge.

Microglia are yolk sac-derived macrophages residing in the parenchyma of brain and spinal cord, where they interact with neurons and other glial. After different conditioning paradigms and bone marrow (BM) or hematopoietic stem cell (HSC) transplantation, graft-derived cells seed the brain and persistently contribute to the parenchymal brain macrophage compartment. Here we establish that graft-derived macrophages acquire, over time, microglia characteristics, including ramified morphology, longevity, radio-resistance and clonal expansion. However, even after prolonged CNS residence, transcriptomes and chromatin accessibility landscapes of engrafted, BM-derived macrophages remain distinct from yolk sac-derived host microglia. Furthermore, engrafted BM-derived cells display discrete responses to peripheral endotoxin challenge, as compared to host microglia. In human HSC transplant recipients, engrafted cells also remain distinct from host microglia, extending our finding to clinical settings. Collectively, our data emphasize the molecular and functional heterogeneity of parenchymal brain macrophages and highlight potential clinical implications for HSC gene therapies aimed to ameliorate lysosomal storage disorders, microgliopathies or general monogenic immuno-deficiencies.

Tumor-specific delivery of therapeutic siRNAs by anti-EGFR immunonanoparticles.

BACKGROUND: Efficient target-specific siRNA delivery has always been a primary concern in the field of siRNA clinical application. PURPOSE: In this study, four different types of anti-epidermal growth factor receptor (EGFR) antibody-conjugated immunonanoparticles were prepared and tested for cancer cell-targeted therapeutic siRNA delivery. MATERIALS AND METHODS: The prepared nanoparticles encapsulating siRNAs were character-ized by gel retardation and particle analysis using a Zetasizer. In vitro transfection and reduction of target genes, vimentin and JAK3, were determined using quantitative reverse transcription polymerase chain reaction. In vivo tumor targeting and antitumoral efficacies of the nanoparticles were evaluated in mice carrying tumors. RESULTS: Among these immunonanoparticles, anti-EGFR immunolipoplexes and immunoviroplexes exhibited remarkable cell binding and siRNA delivery to EGFR-expressing tumor cells compared to immunoliposomes and immunovirosomes. Especially, the anti-EGFR immunoviroplexes exhibited the most efficient siRNA transfection to target tumor cells. Therefore, antitumoral vimentin and Janus kinase-3 siRNAs were loaded in the anti-EGFR immunolipoplexes and immunoviroplexes, which were tested in mice carrying SK-OV-3 tumor xenografts. In fact, the therapeutic siRNAs were efficiently delivered to the tumor tissues by both delivery vehicles, resulting in significant inhibition of tumor growth. Moreover, administration of doxorubicin in combination with anti-EGFR immunoviroplexes resulted in remarkable and synergistic tumor growth inhibition. CONCLUSION: This study provides experimental proof that cancer cell-targeted immunoviroplexes are an efficient siRNA delivery system for cancer therapy. Moreover, this study also suggests that a combination of conventional chemotherapy and tumor-directed anticancer siRNA therapy would be a better modality for cancer treatment.

Safety and Efficacy of Anti-dementia Agents in the Extremely Elderly Patients with Dementia.

BACKGROUND: There are debates on representation and generalizability of previous randomized controlled trials about anti-dementia agents in the oldest old population. In this context, we aimed to investigate the efficacy and safety of anti-dementia agents in the very elderly patients with dementia. METHODS: We conducted a retrospective study of patients with dementia 1) who were 85 years or older, 2) got started anti-dementia agents, and 3) went through follow-up evaluation about one year thereafter. As a control, patients with dementia who were less than 85 years old with similar inclusion criteria were randomly selected during the same period. The adverse drug effects and discontinuation rates were investigated with self-reported complaint after starting or increasing anti-dementia drugs. For efficacy outcome, we also analyzed the change in neuropsychological results during follow-up period. RESULTS: A total of 77 dementia patients who were at least 85 years were enrolled. As a control group, 78 patients with dementia who were younger than 85 was analyzed. The adverse drug effects were observed in 26 (33.3%) patients in the younger old and in 26 (33.8%) in the oldest old (P = 0.095). Twenty-one patients (26.9%) in the younger old group and 13 patients (16.9%) in the oldest old group discontinued their medication (P = 0.131). There were no differences between the two groups about changes of Mini-Mental State Examination and Instrumental Activity of Daily Living scores over time. CONCLUSION: The use of anti-dementia agents in the oldest old dementia patients may be safe and effective as the younger old dementia patients.

Effects of selenium on the survival and invasion of trophoblasts.

OBJECTIVE: Placental oxidative stress is known to be a factor that contributes to pregnancy failure. The aim of this study was to determine whether selenium could induce antioxidant gene expression and regulate invasive activity and mitochondrial activity in trophoblasts, which are a major cell type of the placenta. METHODS: To understand the effects of selenium on trophoblast cells exposed to hypoxia, the viability and invasive activity of trophoblasts were analyzed. The expression of antioxidant enzymes was assessed by reverse-transcription polymerase chain reaction. In addition, the effects of selenium treatment on mitochondrial activity were evaluated in terms of adenosine triphosphate production, mitochondrial membrane potential, and reactive oxygen species levels. RESULTS: Selenium showed positive effects on the viability and migration activity of trophoblast cells when exposed to hypoxia. Interestingly, the increased heme oxygenase 1 expression under hypoxic conditions was decreased by selenium treatment, whereas superoxide dismutase expression was increased in trophoblast cells by selenium treatment for 72 hours, regardless of hypoxia. Selenium-treated trophoblast cells showed increased mitochondrial membrane potential and decreased reactive oxygen species levels under hypoxic conditions for 72 hours. CONCLUSION: These results will be used as basic data for understanding the mechanism of how trophoblast cells respond to oxidative stress and how selenium promotes the upregulation of related genes and improves the survival rate and invasive ability of trophoblasts through regulating mitochondrial activity. These results suggest that selenium may be used in reproductive medicine for purposes including infertility treatment.

Microglial MHC class II is dispensable for experimental autoimmune encephalomyelitis and cuprizone-induced demyelination.

Microglia are resident immune cells in the CNS, strategically positioned to clear dead cells and debris, and orchestrate CNS inflammation and immune defense. In steady state, these macrophages lack MHC class II (MHCII) expression, but microglia activation can be associated with MHCII induction. Whether microglial MHCII serves antigen presentation for critical local T-cell restimulation in CNS auto-immune disorders or modulates microglial signaling output remains under debate. To probe for such scenarios, we generated mice harboring an MHCII deficiency in microglia, but not peripheral myeloid cells. Using the CX3 CR1CreER -based approach we report that microglial antigen presentation is obsolete for the establishment of EAE, with disease onset, progression, and severity unaltered in mutant mice. Antigen presentation-independent roles of microglial MHCII were explored using a demyelination model induced by the copper chelator cuprizone. Absence of microglial I-Ab did not affect the extent of these chemically induced white matter alterations, nor did it affect microglial proliferation or gene expression associated with locally restricted de- and remyelination.

Nanoparticulate vaccine inhibits tumor growth via improved T cell recruitment into melanoma and huHER2 breast cancer.

Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle-immune cell interactions and treatments regime is required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-γ cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction.

Dual-targeting immunoliposomes using angiopep-2 and CD133 antibody for glioblastoma stem cells.

Glioblastoma stem cells (GSCs), which are identified as subpopulation of CD133+/ALDH1+, are known to show resistance to the most of chemotherapy and radiation therapy, leading to the recurrence of tumor in glioblastoma multiforme (GBM) patients. Also, delivery of temozolomide (TMZ), a mainline treatment of GBM, to the GBM site is hampered by various barriers including the blood-brain barrier (BBB). A dual-targeting immunoliposome encapsulating TMZ (Dual-LP-TMZ) was developed by using angiopep-2 (An2) and anti-CD133 monoclonal antibody (CD133 mAb) for BBB transcytosis and specific delivery to GSCs, respectively. The size, zeta potential and drug encapsulation efficiency of Dual-LP-TMZ were 203.4nm in diameter, -1.6mV and 99.2%, respectively. The in vitro cytotoxicity of Dual-LP-TMZ against U87MG GSCs was increased by 425- and 181-folds when compared with that of free TMZ and non-targeted TMZ liposome (LP-TMZ) (10.3µM vs. 4380µM and 1869µM in IC50, respectively). Apoptosis and anti-migration ability of Dual-LP-TMZ in U87MG GSCs were also significantly enhanced comparing with those of free TMZ or LP-TMZ. In vivo study clearly showed a significant reduction in tumor size after intravenous administrations of Dual-LP-TMZ to the orthotopically-implanted brain tumor mice when compared with free TMZ or LP-TMZ. Increased life span (ILS) and median survival time (MST) of tumor-bearing mice were also increased when treated with Dual-LP-TMZ (211.2% in ILS and 49.2days in MST) than with free TMZ (0% in ILS and 23.3day in MST). These data indicate that conjugation of both An2 peptide and CD133 mAb to TMZ-encapsulating liposome is very effective in delivering the TMZ to GSCs via BBB, suggesting a potential use of Dual-LP-TMZ as a therapeutic modality for GBM.

[Small Bowel Perforation Caused by Press-through Package].

With an increased use of the press-through package (PTP) tablet, there has also been an increase in mis-swallowing cases, especially in elderly patients. We report a rare case of PTP-induced small bowel perforation and fistula formation with adjacent small bowel in a healthy elderly patient, who experienced persistent abdominal pain of unknown cause. A 62-year-old healthy man was admitted to our hospital with left abdominal pain that started one month ago. Neither abdominal tenderness nor rebound tenderness was present on physical examination. His vital signs and all other test results were within normal limits. However, a 2.5 cm curved radiopaque material within his thickened small intestine was incidentally detected on an abdominal computed tomography. He underwent laparoscopic small bowel resection, which revealed foreign body in the distal small intestine. Edema, perforation, and adhesions with the surrounding tissues were also noticed in the distal small intestine. Foreign body was turned out to be PTP, and this was considered to be responsible for the small bowel perforation and fistula formation. Precautions regarding PTP usage are necessary to prevent inadvertent PTP ingestion and its related complications, such as perforation, especially in the elderly population.

Loss-of-function screens of druggable targetome against cancer stem-like cells.

Cancer stem-like cells (CSLCs) contribute to the initiation and recurrence of tumors and to their resistance to conventional therapies. In this study, small interfering RNA (siRNA)-based screening of ∼4800 druggable genes in 3-dimensional CSLC cultures in comparison to 2-dimensional bulk cultures of U87 glioma cells revealed 3 groups of genes essential for the following: survival of the CSLC population only, bulk-cultured population only, or both populations. While diverse biologic processes were associated with siRNAs reducing the bulk-cultured population, CSLC-eliminating siRNAs were enriched in a few functional categories, such as lipid metabolism, protein metabolism, and gene expression. Interestingly, siRNAs that selectively reduced CSLC only were found to target genes for cholesterol and unsaturated fatty acid synthesis. The lipidomic profile of CSLCs revealed increased levels of monounsaturated lipids. Pharmacologic blockage of these target pathways reduced CSLCs, and this effect was eliminated by addition of downstream metabolite products. The present CSLC-sensitive target categories provide a useful resource that can be exploited for the selective elimination of CSLCs.-Song, M., Lee, H., Nam, M.-H., Jeong, E., Kim, S., Hong, Y., Kim, N., Yim, H. Y., Yoo, Y.-J., Kim, J. S., Kim, J.-S., Cho, Y.-Y., Mills, G. B., Kim, W.-Y., Yoon, S. Loss-of-function screens of druggable targetome against cancer stem-like cells.