Francesca Zolezzi

C-Myb+ Erythro-Myeloid Progenitor-Derived Fetal Monocytes Give Rise to Adult Tissue-Resident Macrophages

Although classified as hematopoietic cells, tissue-resident macrophages (MFs) arise from embryonic precursors that seed the tissues prior to birth to generate a self-renewing population, which is maintained independently of adult hematopoiesis. Here we reveal the identity of these embryonic precursors using an in utero MF-depletion strategy and fate-mapping of yolk sac (YS) and fetal liver (FL) hematopoiesis. We show that YS MFs are the main precursors of microglia, while most other MFs derive from fetal monocytes (MOs). Both YS MFs and fetal MOs arise from erythro-myeloid progenitors (EMPs) generated in the YS. In the YS, EMPs gave rise to MFs without monocytic intermediates, while EMP seeding the FL upon the establishment of blood circulation acquired c-Myb expression and gave rise to fetal MOs that then seeded embryonic tissues and differentiated into MFs. Thus, adult tissue-resident MFs established from hematopoietic stem cell-independent embryonic precursors arise from two distinct developmental programs.

Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow

Mouse conventional dendritic cells (cDCs) can be classified into two functionally distinct lineages: the CD8α+ (CD103+) cDC1 lineage, and the CD11b+ cDC2 lineage. cDCs arise from a cascade of bone marrow (BM) DC-committed progenitor cells that include the common DC progenitors (CDPs) and pre-DCs, which exit the BM and seed peripheral tissues before differentiating locally into mature cDCs. Where and when commitment to the cDC1 or cDC2 lineage occurs remains poorly understood. Here we found that transcriptional signatures of the cDC1 and cDC2 lineages became evident at the single-cell level from the CDP stage. We also identified Siglec-H and Ly6C as lineage markers that distinguished pre-DC subpopulations committed to the cDC1 lineage (Siglec-H−Ly6C− pre-DCs) or cDC2 lineage (Siglec-H−Ly6C+ pre-DCs). Our results indicate that commitment to the cDC1 or cDC2 lineage occurs in the BM and not in the periphery.

Mapping the human DC lineage through the integration of high-dimensional techniques

Tracing development of the dendritic cell lineage Dendritic cells (DCs) are important components of the immune system that form from the bone marrow into two major cell lineages: plasmacytoid DCs and conventional DCs. See et al. applied single-cell RNA sequencing and cytometry by time-of-flight to characterize the developmental pathways of these cells. They identified blood DC precursors that shared surface markers with plasmacytoid DCs but that were functionally distinct. This unsuspected level of complexity in pre-DC populations reveals additional cell types and refines understanding of known cell types. Science, this issue p. eaag3009 In human blood, the immunological dendritic cell lineage contains many predendritic cell populations. INTRODUCTION Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises multiple subsets, including plasmacytoid DC (pDC) and two functionally specialized lineages of conventional DC (cDC1 and cDC2), whose origins and differentiation pathways remain incompletely defined. RATIONALE As DC are essential regulators of the immune response in health and disease, potential intervention strategies aiming at manipulation of these cells will require in-depth insights of their origins, the mechanisms that govern their homeostasis, and their functional properties. Here, we employed two unbiased high-dimensional technologies to characterize the human DC lineage from bone marrow to blood. RESULTS We isolated the DC-containing population (Lineage−HLA−DR+CD135+ cells) from human blood and defined the transcriptomes of 710 individual cells using massively parallel single-cell mRNA sequencing. By combining complementary bioinformatic approaches, we identified a small cluster of cells within this population as putative DC precursors (pre-DC). We then confirmed this finding using cytometry by time-of-flight (CyTOF) to simultaneously measure the expression of a panel of 38 different proteins at the single-cell level on Lineage−HLA−DR+ cells and found that pre-DC possessed a CD123+CD33+CD45RA+ phenotype. We confirmed the precursor potential of pre-DC by establishing their potential to differentiate in vitro into cDC1 and cDC2, but not pDC, in the known proportions found in vivo. Interestingly, pre-DC also express classical pDC markers, including CD123, CD303, and CD304. Thus, any previous studies using these markers to identify or isolate pDC will have inadvertently included CD123+CD33+ pre-DC. We provide here new markers that can be used to identify unambiguously pre-DC from pDC, including CD33, CX3CR1, CD2, CD5, and CD327. When CD123+CD33+ pre-DC and CD123+CD33− pDC were isolated separately, we observed that pre-DC have unique functional properties that were previously attributed to pDC. Although pDC remain bona fide interferon-α–producing cells, their reported interleukin-12 (IL-12) production and CD4 T cell allostimulatory capacity can likely be attributed to “contaminating” pre-DC. We then asked whether the pre-DC population contained both uncommitted and committed pre-cDC1 and pre-cDC2 precursors, as recently shown in mice. Using microfluidic single-cell mRNA sequencing (scmRNAseq), we showed that the human pre-DC population contains cells exhibiting transcriptomic priming toward cDC1 and cDC2 lineages. Flow cytometry and in vitro DC differentiation experiments further identified CD123+CADM1−CD1c− putative uncommitted pre-DC, alongside CADM1+CD1c− pre-cDC1 and CADM1−CD1c+ pre-cDC2. Finally, we found that pre-DC subsets expressed T cell costimulatory molecules and induced comparable proliferation and polarization of naïve CD4 T cells as adult DC. However, exposure to the Toll-like receptor 9 (TLR9) ligand CpG triggered IL-12p40 and tumor necrosis factor–α production by early pre-DC, pre-cDC1, and pre-cDC2, in contrast to differentiated cDC1 and cDC2, which do not express TLR9. CONCLUSION Using unsupervised scmRNAseq and CyTOF analyses, we have unraveled the complexity of the human DC lineage at the single-cell level, revealing a continuous process of differentiation that starts in the bone marrow (BM) with common DC progenitors (CDP), diverges at the point of emergence of pre-DC and pDC potential, and culminates in maturation of both lineages in the blood and spleen. The pre-DC compartment contains functionally and phenotypically distinct lineage-committed subpopulations, including one early uncommitted CD123+ pre-DC subset and two CD45RA+CD123lo lineage-committed subsets. The discovery of multiple committed pre-DC populations with unique capabilities opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting. Human DC emerge from BM CDP, diverge at the point of emergence of pre-DC and pDC potential, and culminate in maturation of both lineages in the blood. The pre-DC compartment further differentiates into functionally and phenotypically distinct lineage-committed subpopulations, including one early uncommitted CD123+ pre-DC subset (early pre-DC), which give rise to both cDC1 and cDC2 through corresponding CD45RA+CD123lo pre-cDC1 and pre-cDC2 lineage-committed subsets, respectively. Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises two main functionally specialized lineages, whose origins and differentiation pathways remain incompletely defined. Here, we combine two high-dimensional technologies—single-cell messenger RNA sequencing (scmRNAseq) and cytometry by time-of-flight (CyTOF)—to identify human blood CD123+CD33+CD45RA+ DC precursors (pre-DC). Pre-DC share surface markers with plasmacytoid DC (pDC) but have distinct functional properties that were previously attributed to pDC. Tracing the differentiation of DC from the bone marrow to the peripheral blood revealed that the pre-DC compartment contains distinct lineage-committed subpopulations, including one early uncommitted CD123high pre-DC subset and two CD45RA+CD123low lineage-committed subsets exhibiting functional differences. The discovery of multiple committed pre-DC populations opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.

Rational Design of a Live Attenuated Dengue Vaccine: 2 '-O-Methyltransferase Mutants Are Highly Attenuated and Immunogenic in Mice and Macaques

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2′-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2′-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2′-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the hosts innate immune response.

Migratory, and not lymphoid-resident, dendritic cells maintain peripheral self-tolerance and prevent autoimmunity via induction of iTreg cells

There is evidence that dendritic cells (DCs) induce peripheral tolerance. Nevertheless, it is not known whether immature DCs in general are able to tolerize CD4(+) T cells or if this is a prerogative of specialized subtypes. Here we show that, when autoantigen presentation is extended to all conventional mouse DCs, immature lymphoid tissue resident DCs are unable to induce autoantigen-specific regulatory T (iTreg) cell conversion. In contrast, this is an exclusive prerogative of steady-state migratory DCs. Because only lymph nodes host migratory DCs, iTreg cells develop and are retained solely in lymph nodes, and not in the spleen. Mechanistically, in cutaneous lymph nodes, DC-derived CCL22 contributes to the retention of iTreg cells. The importance of the local generation of iTreg cells is emphasized by their essential role in preventing autoimmunity.

Intestinal CD103(+)CD11b(-) dendritic cells restrain colitis via IFN-γ-induced anti-inflammatory response in epithelial cells.

A crosstalk between commensals, gut immune cells, and colonic epithelia is required for a proper function of intestinal mucosal barrier. Here we investigated the importance of two distinct intestinal dendritic cell (DC) subsets in controlling intestinal inflammation. We show that Clec9A–diphtheria toxin receptor (DTR) mice after depletion of CD103+CD11b− DCs developed severe, low-dose dextran sodium sulfate (DSS)-induced colitis, whereas the lack of CD103+CD11b+ DCs in Clec4a4-DTR mice did not exacerbate intestinal inflammation. The CD103+CD11b− DC subset has gained a functional specialization that able them to repress inflammation via several epithelial interferon-γ (IFN-γ)-induced proteins. Among others, we identified that epithelial IDO1 and interleukin-18-binding protein (IL-18bp) were strongly modulated by CD103+CD11b− DCs. Through its preferential property to express IL-12 and IL-15, this particular DC subset can induce lymphocytes in colonic lamina propria and in epithelia to secrete IFN-γ that then can trigger a reversible early anti-inflammatory response in intestinal epithelial cells.

CD103+ Dendritic Cells Control Th17 Cell Function in the Lung

Th17 cells express diverse functional programs while retaining their Th17 identity, in some cases exhibiting a stem-cell-like phenotype. Whereas the importance of Th17 cell regulation in autoimmune and infectious diseases is firmly established, the signaling pathways controlling their plasticity are undefined. Using a mouse model of invasive pulmonary aspergillosis, we found that lung CD103(+) dendritic cells (DCs) would produce IL-2, dependent on NFAT signaling, leading to an optimally protective Th17 response. The absence of IL-2 in DCs caused unrestrained production of IL-23 and fatal hyperinflammation, which was characterized by strong Th17 polarization and the emergence of a Th17 stem-cell-like population. Although several cell types may be affected by deficient IL-2 production in DCs, our findings identify the balance between IL-2 and IL-23 productions by lung DCs as an important regulator of the local inflammatory response to infection.

Mutant SOD1 and mitochondrial damage alter expression and splicing of genes controlling neuritogenesis in models of neurodegeneration

Mitochondrial dysfunction has been implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson, Alzheimer, and Amyotrophic Lateral Sclerosis (ALS). In addition, aberrant mRNA splicing has been documented in neurodegeneration. To characterize the cellular response to mitochondrial perturbations at the level of gene expression and alternative pre‐mRNA splicing we used splicing‐sensitive microarrays to profile human neuroblastoma SH‐SY5Y cells treated with paraquat, a neurotoxic herbicide that induces the formation of reactive oxygen species and causes mitochondrial damage in animal models, and SH‐SY5Y cells stably expressing the mutant G93A‐SOD1 protein, one of the genetic causes of ALS. In both models we identified a common set of genes whose expression and alternative splicing are deregulated. Pathway analysis of the deregulated genes revealed enrichment in genes involved in neuritogenesis, axon growth and guidance, and synaptogenesis. Alterations in transcription and pre‐mRNA splicing of candidate genes were confirmed experimentally in the cell line models as well as in brain and spinal cord of transgenic mice carrying the G93A‐SOD1 mutation. Our findings expand the realm of the pathways implicated in neurodegeneration and suggest that alterations of axonal function may descend directly from mitochondrial damage. Hum Mutat 31:1–15, 2011.

Cell Specific eQTL Analysis without Sorting Cells

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn’s disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.

Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation through CXCL2

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation, and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine and/or paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.