Meningeal macrophages protect against viral neuroinfection.

The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II- MM were abundant neonatally, whereas MHC-II+ MM appeared over time. These barrier macrophages differentially responded to in vivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II+ MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation.

Broad and Largely Concordant Molecular Changes Characterize Tolerogenic and Immunogenic Dendritic Cell Maturation in Thymus and Periphery.

Dendritic cells (DCs) are instrumental in the initiation of T cell responses, but how thymic and peripheral tolerogenic DCs differ globally from Toll-like receptor (TLR)-induced immunogenic DCs remains unclear. Here, we show that thymic XCR1(+) DCs undergo a high rate of maturation, accompanied by profound gene-expression changes that are essential for central tolerance and also happen in germ-free mice. Those changes largely overlap those occurring during tolerogenic and, more unexpectedly, TLR-induced maturation of peripheral XCR1(+) DCs, arguing against the commonly held view that tolerogenic DCs undergo incomplete maturation. Interferon-stimulated gene (ISG) expression was among the few discriminators of immunogenic and tolerogenic XCR1(+) DCs. Tolerogenic XCR1(+) thymic DCs were, however, unique in expressing ISGs known to restrain virus replication. Therefore, a broad functional convergence characterizes tolerogenic and immunogenic XCR1(+) DC maturation in the thymus and periphery, maximizing antigen presentation and signal delivery to developing and to conventional and regulatory mature T cells.

An Slfn2 mutation causes lymphoid and myeloid immunodeficiency due to loss of immune cell quiescence.

Here we describe a previously unknown form of inherited immunodeficiency revealed by an N-ethyl-N-nitrosourea-induced mutation called elektra. Mice homozygous for this mutation showed enhanced susceptibility to bacterial and viral infection and diminished numbers of T cells and inflammatory monocytes that failed to proliferate after infection and died via the intrinsic apoptotic pathway in response to diverse proliferative stimuli. They also had a greater proportion of T cells poised to replicate DNA, and their T cells expressed a subset of activation markers, suggestive of a semi-activated state. We positionally ascribe the elektra phenotype to a mutation in the gene encoding Schlafen-2 (Slfn2). Our findings identify a physiological role for Slfn2 in the defense against pathogens through the regulation of quiescence in T cells and monocytes.

Natural Killer Cell Sensing of Infected Cells Compensates for MyD88 Deficiency but Not IFN-I Activity in Resistance to Mouse Cytomegalovirus.

In mice, plasmacytoid dendritic cells (pDC) and natural killer (NK) cells both contribute to resistance to systemic infections with herpes viruses including mouse Cytomegalovirus (MCMV). pDCs are the major source of type I IFN (IFN-I) during MCMV infection. This response requires pDC-intrinsic MyD88-dependent signaling by Toll-Like Receptors 7 and 9. Provided that they express appropriate recognition receptors such as Ly49H, NK cells can directly sense and kill MCMV-infected cells. The loss of any one of these responses increases susceptibility to infection. However, the relative importance of these antiviral immune responses and how they are related remain unclear. In humans, while IFN-I responses are essential, MyD88 is dispensable for antiviral immunity. Hence, a higher redundancy has been proposed in the mechanisms promoting protective immune responses against systemic infections by herpes viruses during natural infections in humans. It has been assumed, but not proven, that mice fail to mount protective MyD88-independent IFN-I responses. In humans, the mechanism that compensates MyD88 deficiency has not been elucidated. To address these issues, we compared resistance to MCMV infection and immune responses between mouse strains deficient for MyD88, the IFN-I receptor and/or Ly49H. We show that selective depletion of pDC or genetic deficiencies for MyD88 or TLR9 drastically decreased production of IFN-I, but not the protective antiviral responses. Moreover, MyD88, but not IFN-I receptor, deficiency could largely be compensated by Ly49H-mediated antiviral NK cell responses. Thus, contrary to the current dogma but consistent with the situation in humans, we conclude that, in mice, in our experimental settings, MyD88 is redundant for IFN-I responses and overall defense against a systemic herpes virus infection. Moreover, we identified direct NK cell sensing of infected cells as one mechanism able to compensate for MyD88 deficiency in mice. Similar mechanisms likely contribute to protect MyD88- or IRAK4-deficient patients from viral infections.

Plasmacytoid, conventional, and monocyte-derived dendritic cells undergo a profound and convergent genetic reprogramming during their maturation.

DCs express receptors sensing microbial, danger or cytokine signals, which when triggered in combination drive DC maturation and functional polarization. Maturation was proposed to result from a discrete number of modifications in conventional DCs (cDCs), in contrast to a cell-fate conversion in plasmacytoid DCs (pDCs). cDC maturation is generally assessed by measuring cytokine production and membrane expression of MHC class II and co-stimulation molecules. pDC maturation complexity was demonstrated by functional genomics. Here, pDCs and cDCs were shown to undergo profound and convergent changes in their gene expression programs in vivo during viral infection. This observation was generalized to other stimulation conditions and DC subsets, by public microarray data analyses, PCR confirmation of selected gene expression profiles, and gene regulatory sequence bioinformatics analyses. Thus, maturation is a complex process similarly reshaping all DC subsets, including through the induction of a core set of NF-κB- or IFN-stimulated genes irrespective of stimuli.

Comparative genomics as a tool to reveal functional equivalences between human and mouse dendritic cell subsets.

During evolution, vertebrates have developed an adaptive immune system able to cope with a variety of pathogens. Dendritic cells (DCs) are central to this process. DCs integrate information derived from pathogens or endogenous danger signals and convey them to T lymphocytes. Most of the present knowledge on DCs was generated in mice or by using human DCs differentiated in vitro from monocytes. In both species, several DC subsets have been identified in vivo based on differences in their phenotypes, anatomical locations or functions. In mice, protective immunity against intracellular pathogens or tumors can be induced most efficiently by targeting antigens to the CD8 alpha(+) DCs, a subset of DCs which resides in lymphoid tissues and is especially efficient at cross-presenting exogenous antigens to CD8(+) T lymphocytes. In contrary, harnessing human DC subsets for medical purposes is currently hampered by insufficient knowledge about these cells. To overcome this cognitive gap, we are using comparative genomics as a tool for designing hypotheses and experiments to further characterize DC subset functions and their molecular control, including the investigation of the functional equivalences that might exist between human and mouse DC subsets.

Jinx, an MCMV susceptibility phenotype caused by disruption of Unc13d: a mouse model of type 3 familial hemophagocytic lymphohistiocytosis.

Mouse cytomegalovirus (MCMV) susceptibility often results from defects of natural killer (NK) cell function. Here we describe Jinx, an N-ethyl-N-nitrosourea-induced MCMV susceptibility mutation that permits unchecked proliferation of the virus, causing death. In Jinx homozygotes, activated NK cells and cytotoxic T lymphocytes (CTLs) fail to degranulate, although they retain the ability to produce cytokines, and cytokine levels are markedly elevated in the blood of infected mutant mice. Jinx was mapped to mouse chromosome 11 on a total of 246 meioses and confined to a 4.60-million basepair critical region encompassing 122 annotated genes. The phenotype was ascribed to the creation of a novel donor splice site in Unc13d, the mouse orthologue of human MUNC13-4, in which mutations cause type 3 familial hemophagocytic lymphohistiocytosis (FHL3), a fatal disease marked by massive hepatosplenomegaly, anemia, and thrombocytopenia. Jinx mice do not spontaneously develop clinical features of hemophagocytic lymphohistiocytosis (HLH), but do so when infected with lymphocytic choriomeningitis virus, exhibiting hyperactivation of CTLs and antigen-presenting cells, and inadequate restriction of viral proliferation. In contrast, neither Listeria monocytogenes nor MCMV induces the syndrome. In mice, the HLH phenotype is conditional, which suggests the existence of a specific infectious trigger of FHL3 in humans.

Inflammatory monocytes and neutrophils are licensed to kill during memory responses in vivo.

Immunological memory is a hallmark of B and T lymphocytes that have undergone a previous encounter with a given antigen. It is assumed that memory cells mediate better protection of the host upon re-infection because of improved effector functions such as antibody production, cytotoxic activity and cytokine secretion. In contrast to cells of the adaptive immune system, innate immune cells are believed to exhibit a comparable functional effector response each time the same pathogen is encountered. Here, using mice infected by the intracellular bacterium Listeria monocytogenes, we show that during a recall bacterial infection, the chemokine CCL3 secreted by memory CD8+ T cells drives drastic modifications of the functional properties of several populations of phagocytes. We found that inflammatory ly6C+ monocytes and neutrophils largely mediated memory CD8+ T cell bacteriocidal activity by producing increased levels of reactive oxygen species (ROS), augmenting the pH of their phagosomes and inducing antimicrobial autophagy. These events allowed an extremely rapid control of bacterial growth in vivo and accounted for protective immunity. Therefore, our results provide evidence that cytotoxic memory CD8+ T cells can license distinct antimicrobial effector mechanisms of innate cells to efficiently clear pathogens.

Disruption of MyD88 signaling suppresses hemophagocytic lymphohistiocytosis in mice.

Hemophagocytic lymphohistiocytosis (HLH) is a rare inflammatory disorder with a poor prognosis for affected individuals. To find a means of suppressing the clinical phenotype, we investigated the cellular and molecular mechanisms leading to HLH in Unc13d(jinx/jinx) mice, in which cytolytic function of NK and CD8(+) T cells is impaired. Unc13d(jinx/jinx) mutants infected with lymphochoriomeningitis virus (LCMV) present typical clinical features of HLH, including splenomegaly, elevated serum IFNγ, and anemia. Proteins mediating cell-cell contact, cytokine signaling or Toll-like receptor (TLR) signaling were analyzed. We show that neither the integrin CD18, which is involved in adhesion between antigen-presenting cells and effector T cells, nor tumor necrosis factor (TNF) made nonredundant contributions to the disease phenotype. Disruption of IFNγ signaling reduced immune cell activation in Unc13d(jinx/jinx) mice, but also resulted in uncontrolled viral proliferation and exaggerated release of inflammatory cytokines. Abrogating the function of myeloid differentiation primary response gene 88 (MyD88) in Unc13d(jinx/jinx) mice suppressed immune cell activation and controlled cytokine production in an IL-1 receptor 1 (IL-1R1)-independent way. Our findings implicate MyD88 as the key initiator of myeloid and lymphoid proliferation in HLH, and suggest that blockade of this signaling molecule may reduce immunopathology in patients.

Impact of ß2 integrin deficiency on mouse natural killer cell development and function.

Natural killer (NK) cells are innate immune cells that express members of the leukocyte ß2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that ß2 integrin-deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit(+) cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, ß2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

Cutting edge: expression of XCR1 defines mouse lymphoid-tissue resident and migratory dendritic cells of the CD8α+ type.

Subsets of dendritic cells (DCs) have been described according to their functions and anatomical locations. Conventional DC subsets are defined by reciprocal expression of CD11b and CD8α in lymphoid tissues (LT), and of CD11b and CD103 in non-LT (NLT). Spleen CD8α(+) and dermal CD103(+) DCs share a high efficiency for Ag cross-presentation and a developmental dependency on specific transcription factors. However, it is not known whether all NLT-derived CD103(+) DCs and LT-resident CD8α(+) DCs are similar despite their different anatomical locations. XCR1 was previously described as exclusively expressed on mouse spleen CD8α(+) DCs and human blood BDCA3(+) DCs. In this article, we showed that LT-resident CD8α(+) DCs and NLT-derived CD103(+) DCs specifically express XCR1 and are characterized by a unique transcriptional fingerprint, irrespective of their tissue of origin. Therefore, CD8α(+) DCs and CD103(+) DCs belong to a common DC subset which is unequivocally identified by XCR1 expression throughout the body.

Flt3 permits survival during infection by rendering dendritic cells competent to activate NK cells.

A previously unappreciated signal necessary for dendritic cell (DC)-mediated activation of natural killer (NK) cells during viral infection was revealed by a recessive N-ethyl-N-nitrosourea-induced mutation called warmflash (wmfl). Wmfl homozygotes displayed increased susceptibility to mouse cytomegalovirus (MCMV) infection. In response to MCMV infection in vivo, delayed NK cell activation was observed, but no intrinsic defects in NK cell activation or function were identified. Rather, coculture experiments demonstrated that NK cells are suboptimally activated by wmfl DCs, which showed impaired cytokine production in response to MCMV or synthetic TLR7 and TLR9 ligands. The wmfl mutation was identified in the gene encoding the Fms-like tyrosine kinase 3 (Flt3). Flt3 ligand (Flt3L) is transiently induced in the serum upon infection or TLR activation. However, antibody blockade reveals no acute requirement for Flt3L, suggesting that the Flt3L --> Flt3 axis programs the development of DCs, making them competent to support NK effector function. In the absence of Flt3 signaling, NK cell activation is delayed and survival during MCMV infection is markedly compromised.

Crosstalk between components of the innate immune system: promoting anti-microbial defenses and avoiding immunopathologies.

Because it reaches full functional efficacy rapidly upon encounter with a pathogen, the innate immune system is considered as the first line of defense against infections. The sensing of microbes or of transformed or infected cells, through innate immune recognition receptors (referred to as activating I2R2), initiates pro-inflammatory responses and innate immune effector functions. Other I2R2 with inhibitory properties bind self-ligands constitutively expressed in host. However, this dichotomy in the recognition of foreign or induced self versus constitutive self by I2R2 is not always respected in certain non-infectious conditions reminiscent of immunopathologies. In this review, we discuss that immune mechanisms have evolved to avoid inappropriate inflammatory disorders in individuals. Molecular crossregulation exists between components of I2R2 signaling pathways, and intricate interactions between cells from both innate and adaptive immune systems set the bases of controlled immune responses. We also pinpoint that, like T or B cells, some cells of the innate immune system must go through education processes to prevent autoreactivity. In addition, we illustrate how gene expression profiling of immune cell types is a useful tool to find functional homologies between cell subsets of different species and to speculate about unidentified functions of these cells in the responses to pathogen infections.

Network analysis of large-scale ImmGen and Tabula Muris datasets highlights metabolic diversity of tissue mononuclear phagocytes.

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages.

Skin-draining lymph nodes contain dermis-derived CD103(-) dendritic cells that constitutively produce retinoic acid and induce Foxp3(+) regulatory T cells.

Small intestinal CD103(+) dendritic cells (DCs) have the selective ability to promote de novo generation of regulatory T cells via the production of retinoic acid (RA). Considering that aldehyde dehydrogenase (ALDH) activity controls the production of RA, we used a flow cytometry-based assay to measure ALDH activity at the single-cell level and to perform a comprehensive analysis of the RA-producing DC populations present in lymphoid and nonlymphoid mouse tissues. RA-producing DCs were primarily of the tissue-derived, migratory DC subtype and can be readily found in the skin and in the lungs as well as in their corresponding draining lymph nodes. The RA-producing skin-derived DCs were capable of triggering the generation of regulatory T cells, a finding demonstrating that the presence of RA-producing, tolerogenic DCs is not restricted to the intestinal tract as previously thought. Unexpectedly, the production of RA by skin DCs was restricted to CD103(-) DCs, indicating that CD103 expression does not constitute a "universal" marker for RA-producing mouse DCs. Finally, Toll-like receptor (TLR) triggering or the presence of a commensal microflora was not essential for the induction of ALDH activity in the discrete ALDH(+) DC subsets that characterize tissues constituting environmental interfaces.

Commitment to the regulatory T cell lineage requires CARMA1 in the thymus but not in the periphery.

Regulatory T (T(reg)) cells expressing forkhead box P3 (Foxp3) arise during thymic selection among thymocytes with modestly self-reactive T cell receptors. In vitro studies suggest Foxp3 can also be induced among peripheral CD4(+) T cells in a cytokine dependent manner. T(reg) cells of thymic or peripheral origin may serve different functions in vivo, but both populations are phenotypically indistinguishable in wild-type mice. Here we show that mice with a Carma1 point mutation lack thymic CD4(+)Foxp3(+) T(reg) cells and demonstrate a cell-intrinsic requirement for CARMA1 in thymic Foxp3 induction. However, peripheral Carma1-deficient T(reg) cells could be generated and expanded in vitro in response to the cytokines transforming growth factor beta (TGFbeta) and interleukin-2 (IL-2). In vivo, a small peripheral T(reg) pool existed that was enriched at mucosal sites and could expand systemically after infection with mouse cytomegalovirus (MCMV). Our data provide genetic evidence for two distinct mechanisms controlling regulatory T cell lineage commitment. Furthermore, we show that peripheral T(reg) cells are a dynamic population that may expand to limit immunopathology or promote chronic infection.

Existence of CD8α-like dendritic cells with a conserved functional specialization and a common molecular signature in distant mammalian species.

The mouse lymphoid organ-resident CD8alpha(+) dendritic cell (DC) subset is specialized in Ag presentation to CD8(+) T cells. Recent evidence shows that mouse nonlymphoid tissue CD103(+) DCs and human blood DC Ag 3(+) DCs share similarities with CD8alpha(+) DCs. We address here whether the organization of DC subsets is conserved across mammals in terms of gene expression signatures, phenotypic characteristics, and functional specialization, independently of the tissue of origin. We study the DC subsets that migrate from the skin in the ovine species that, like all domestic animals, belongs to the Laurasiatheria, a distinct phylogenetic clade from the supraprimates (human/mouse). We demonstrate that the minor sheep CD26(+) skin lymph DC subset shares significant transcriptomic similarities with mouse CD8alpha(+) and human blood DC Ag 3(+) DCs. This allowed the identification of a common set of phenotypic characteristics for CD8alpha-like DCs in the three mammalian species (i.e., SIRP(lo), CADM1(hi), CLEC9A(hi), CD205(hi), XCR1(hi)). Compared to CD26(-) DCs, the sheep CD26(+) DCs show 1) potent stimulation of allogeneic naive CD8(+) T cells with high selective induction of the Ifngamma and Il22 genes; 2) dominant efficacy in activating specific CD8(+) T cells against exogenous soluble Ag; and 3) selective expression of functional pathways associated with high capacity for Ag cross-presentation. Our results unravel a unifying definition of the CD8alpha(+)-like DCs across mammalian species and identify molecular candidates that could be used for the design of vaccines applying to mammals in general.

Mice with mutations of Dock7 have generalized hypopigmentation and white-spotting but show normal neurological function.

The classical recessive coat color mutation misty (m) arose spontaneously on the DBA/J background and causes generalized hypopigmentation and localized white-spotting in mice, with a lack of pigment on the belly, tail tip, and paws. Here we describe moonlight (mnlt), a second hypopigmentation and white-spotting mutation identified on the C57BL/6J background, which yields a phenotypic copy of m/m coat color traits. We demonstrate that the 2 mutations are allelic. m/m and mnlt/mnlt phenotypes both result from mutations that truncate the dedicator of cytokinesis 7 protein (DOCK7), a widely expressed Rho family guanine nucleotide exchange factor. Although Dock7 is transcribed at high levels in the developing brain and has been implicated in both axon development and myelination by in vitro studies, we find no requirement for DOCK7 in neurobehavioral function in vivo. However, DOCK7 has non-redundant role(s) related to the distribution and function of dermal and follicular melanocytes.

CD36 is a sensor of diacylglycerides.

Toll-like receptor 2 (TLR2) is required for the recognition of numerous molecular components of bacteria, fungi and protozoa. The breadth of the ligand repertoire seems unusual, even if one considers that TLR2 may form heteromers with TLRs 1 and 6 (ref. 12), and it is likely that additional proteins serve as adapters for TLR2 activation. Here we show that an N-ethyl-N-nitrosourea-induced nonsense mutation of Cd36 (oblivious) causes a recessive immunodeficiency phenotype in which macrophages are insensitive to the R-enantiomer of MALP-2 (a diacylated bacterial lipopeptide) and to lipoteichoic acid. Homozygous mice are hypersusceptible to Staphylococcus aureus infection. Cd36(obl) macrophages readily detect S-MALP-2, PAM(2)CSK(4), PAM(3)CSK(4) and zymosan, revealing that some--but not all--TLR2 ligands are dependent on CD36. Already known as a receptor for endogenous molecules, CD36 is also a selective and nonredundant sensor of microbial diacylglycerides that signal via the TLR2/6 heterodimer.

Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8+ T cells.

Successful immune responses rely on a regulated delivery of the right signals to the right cells at the right time. Here we show that natural killer (NK) and dendritic epidermal γδ T cells (DETCs) use similar mechanisms to spatiotemporally orchestrate conventional type 1 dendritic cell (cDC1) functions in the spleen, skin, and its draining lymph nodes (dLNs). Upon MCMV infection in the spleen, cDC1 clusterize with activated NK cells in marginal zones. This XCR1-dependent repositioning of cDC1 toward NK cells allows contact delivery of IL-12 and IL-15/IL-15Rα by cDC1, which is critical for NK cell responses. NK cells deliver granulocyte-macrophage colony-stimulating factor (GM-CSF) to cDC1, guiding their CCR7-dependent relocalization into the T cell zone. In MCMV-infected skin, XCL1-secreting DETCs promote cDC1 migration from the skin to the dLNs. This XCR1-dependent licensing of cDC1 both in the spleen and skin accelerates antiviral CD8+ T cell responses, revealing an additional mechanism through which cDC1 bridge innate and adaptive immunity.