The Phytophthora parasitica effector AVH195 interacts with ATG8, attenuates host autophagy, and promotes biotrophic infection.

BACKGROUND: Plant pathogens secrete effector proteins into host cells to suppress immune responses and manipulate fundamental cellular processes. One of these processes is autophagy, an essential recycling mechanism in eukaryotic cells that coordinates the turnover of cellular components and contributes to the decision on cell death or survival. RESULTS: We report the characterization of AVH195, an effector from the broad-spectrum oomycete plant pathogen, Phytophthora parasitica. We show that P. parasitica expresses AVH195 during the biotrophic phase of plant infection, i.e., the initial phase in which host cells are maintained alive. In tobacco, the effector prevents the initiation of cell death, which is caused by two pathogen-derived effectors and the proapoptotic BAX protein. AVH195 associates with the plant vacuolar membrane system and interacts with Autophagy-related protein 8 (ATG8) isoforms/paralogs. When expressed in cells from the green alga, Chlamydomonas reinhardtii, the effector delays vacuolar fusion and cargo turnover upon stimulation of autophagy, but does not affect algal viability. In Arabidopsis thaliana, AVH195 delays the turnover of ATG8 from endomembranes and promotes plant susceptibility to P. parasitica and the obligate biotrophic oomycete pathogen Hyaloperonospora arabidopsidis. CONCLUSIONS: Taken together, our observations suggest that AVH195 targets ATG8 to attenuate autophagy and prevent associated host cell death, thereby favoring biotrophy during the early stages of the infection process.

Agonist-induced functional analysis and cell sorting associated with single-cell transcriptomics characterizes cell subtypes in normal and pathological brain.

To gain better insight into the dynamic interaction between cells and their environment, we developed the agonist-induced functional analysis and cell sorting (aiFACS) technique, which allows the simultaneous recording and sorting of cells in real-time according to their immediate and individual response to a stimulus. By modulating the aiFACS selection parameters, testing different developmental times, using various stimuli, and multiplying the analysis of readouts, it is possible to analyze cell populations of any normal or pathological tissue. The association of aiFACS with single-cell transcriptomics allows the construction of functional tissue cartography based on specific pharmacological responses of cells. As a proof of concept, we used aiFACS on the dissociated mouse brain, a highly heterogeneous tissue, enriching it in interneurons by stimulation with KCl or with AMPA, an agonist of the glutamate receptors, followed by sorting based on calcium levels. After AMPA stimulus, single-cell transcriptomics of these aiFACS-selected interneurons resulted in a nine-cluster classification. Furthermore, we used aiFACS on interneurons derived from the brain of the Fmr1-KO mouse, a rodent model of fragile X syndrome. We showed that these interneurons manifest a generalized defective response to AMPA compared with wild-type cells, affecting all the analyzed cell clusters at one specific postnatal developmental time.

Accumulation of amyloid precursor protein C-terminal fragments triggers mitochondrial structure, function, and mitophagy defects in Alzheimer's disease models and human brains.

Several lines of recent evidence indicate that the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) could correspond to an etiological trigger of Alzheimer's disease (AD) pathology. Altered mitochondrial homeostasis is considered an early event in AD development. However, the specific contribution of APP-CTFs to mitochondrial structure, function, and mitophagy defects remains to be established. Here, we demonstrate in neuroblastoma SH-SY5Y cells expressing either APP Swedish mutations, or the ß-secretase-derived APP-CTF fragment (C99) combined with ß- and γ-secretase inhibition, that APP-CTFs accumulation independently of Aß triggers excessive mitochondrial morphology alteration (i.e., size alteration and cristae disorganization) associated with enhanced mitochondrial reactive oxygen species production. APP-CTFs accumulation also elicit basal mitophagy failure illustrated by enhanced conversion of LC3, accumulation of LC3-I and/or LC3-II, non-degradation of SQSTM1/p62, inconsistent Parkin and PINK1 recruitment to mitochondria, enhanced levels of membrane and matrix mitochondrial proteins, and deficient fusion of mitochondria with lysosomes. We confirm the contribution of APP-CTFs accumulation to morphological mitochondria alteration and impaired basal mitophagy in vivo in young 3xTgAD transgenic mice treated with γ-secretase inhibitor as well as in adeno-associated-virus-C99 injected mice. Comparison of aged 2xTgAD and 3xTgAD mice indicates that, besides APP-CTFs, an additional contribution of Aß to late-stage mitophagy activation occurs. Importantly, we report on mitochondrial accumulation of APP-CTFs in human post-mortem sporadic AD brains correlating with mitophagy failure molecular signature. Since defective mitochondria homeostasis plays a pivotal role in AD pathogenesis, targeting mitochondrial dysfunctions and/or mitophagy by counteracting early APP-CTFs accumulation may represent relevant therapeutic interventions in AD.

Hybridization and polyploidy enable genomic plasticity without sex in the most devastating plant-parasitic nematodes.

Root-knot nematodes (genus Meloidogyne) exhibit a diversity of reproductive modes ranging from obligatory sexual to fully asexual reproduction. Intriguingly, the most widespread and devastating species to global agriculture are those that reproduce asexually, without meiosis. To disentangle this surprising parasitic success despite the absence of sex and genetic exchanges, we have sequenced and assembled the genomes of three obligatory ameiotic and asexual Meloidogyne. We have compared them to those of relatives able to perform meiosis and sexual reproduction. We show that the genomes of ameiotic asexual Meloidogyne are large, polyploid and made of duplicated regions with a high within-species average nucleotide divergence of ~8%. Phylogenomic analysis of the genes present in these duplicated regions suggests that they originated from multiple hybridization events and are thus homoeologs. We found that up to 22% of homoeologous gene pairs were under positive selection and these genes covered a wide spectrum of predicted functional categories. To biologically assess functional divergence, we compared expression patterns of homoeologous gene pairs across developmental life stages using an RNAseq approach in the most economically important asexually-reproducing nematode. We showed that >60% of homoeologous gene pairs display diverged expression patterns. These results suggest a substantial functional impact of the genome structure. Contrasting with high within-species nuclear genome divergence, mitochondrial genome divergence between the three ameiotic asexuals was very low, signifying that these putative hybrids share a recent common maternal ancestor. Transposable elements (TE) cover a ~1.7 times higher proportion of the genomes of the ameiotic asexual Meloidogyne compared to the sexual relative and might also participate in their plasticity. The intriguing parasitic success of asexually-reproducing Meloidogyne species could be partly explained by their TE-rich composite genomes, resulting from allopolyploidization events, and promoting plasticity and functional divergence between gene copies in the absence of sex and meiosis.

The "one airway, one disease" concept in light of Th2 inflammation.

In line with the pathophysiological continuum described between nose and bronchus in allergic respiratory diseases, we assessed whether nasal epithelium could mirror the Type 2 T-helper cell (Th2) status of bronchial epithelium.Nasal and bronchial cells were collected by brushing from healthy controls (C, n=13), patients with allergic rhinitis and asthma (AR, n=12), and patients with isolated allergic rhinitis (R, n=14). Cellular composition was assessed by flow cytometry, gene expression was analysed by RNA sequencing and Th2, Type 17 T-helper cell (Th17) and interferon (IFN) signatures were derived from the literature.Infiltration by polymorphonuclear neutrophils (PMN) in the nose excluded 30% of the initial cohort. All bronchial samples from the AR group were Th2-high. The gene expression profile of nasal samples from the AR group correctly predicted the paired bronchial sample Th2 status in 71% of cases. Nevertheless, nasal cells did not appear to be a reliable surrogate for the Th2 response, in particular due to a more robust influence of the IFN response in 14 out of 26 nasal samples. The Th2 scores in the nose and bronchi correlated with mast cell count (both p<0.001) and number of sensitisations (p=0.006 and 0.002), while the Th17 scores correlated with PMN count (p=0.006 and 0.003).The large variability in nasal cell composition and type of inflammation restricts its use as a surrogate for assessing bronchial Th2 inflammation in AR patients.

CD8+ T cells are essential for the effects of enriched environment on hippocampus-dependent behavior, hippocampal neurogenesis and synaptic plasticity.

Enriched environment (EE) induces plasticity changes in the brain. Recently, CD4+ T cells have been shown to be involved in brain plasticity processes. Here, we show that CD8+ T cells are required for EE-induced brain plasticity in mice, as revealed by measurements of hippocampal volume, neurogenesis in the DG of the hippocampus, spinogenesis and glutamatergic synaptic function in the CA of the hippocampus. As a consequence, EE-induced behavioral benefits depend, at least in part, on CD8+ T cells. In addition, we show that spleen CD8+ T cells from mice housed in standard environment (SE) and EE have different properties in terms of 1) TNFα release after in vitro CD3/CD28 or PMA/Iono stimulation 2) in vitro proliferation properties 3) CD8+ CD44+ CD62Llow and CD62Lhi T cells repartition 4) transcriptomic signature as revealed by RNA sequencing. CD8+ T cells purified from the choroid plexus of SE and EE mice also exhibit different transcriptomic profiles as highlighted by single-cell mRNA sequencing. We show that CD8+ T cells are essential mediators of beneficial EE effects on brain plasticity and cognition. Additionally, we propose that EE differentially primes CD8+ T cells leading to behavioral improvement.

Central CCL2 signaling onto MCH neurons mediates metabolic and behavioral adaptation to inflammation.

Sickness behavior defines the endocrine, autonomic, behavioral, and metabolic responses associated with infection. While inflammatory responses were suggested to be instrumental in the loss of appetite and body weight, the molecular underpinning remains unknown. Here, we show that systemic or central lipopolysaccharide (LPS) injection results in specific hypothalamic changes characterized by a precocious increase in the chemokine ligand 2 (CCL2) followed by an increase in pro-inflammatory cytokines and a decrease in the orexigenic neuropeptide melanin-concentrating hormone (MCH). We therefore hypothesized that CCL2 could be the central relay for the loss in body weight induced by the inflammatory signal LPS. We find that central delivery of CCL2 promotes neuroinflammation and the decrease in MCH and body weight. MCH neurons express CCL2 receptor and respond to CCL2 by decreasing both electrical activity and MCH release. Pharmacological or genetic inhibition of CCL2 signaling opposes the response to LPS at both molecular and physiologic levels. We conclude that CCL2 signaling onto MCH neurons represents a core mechanism that relays peripheral inflammation to sickness behavior.

Exploiting cell cycle inhibitor genes of the KRP family to control root-knot nematode induced feeding sites in plants.

Cell cycle control in galls provoked by root-knot nematodes involves the activity of inhibitor genes like the Arabidopsis ICK/KRP members. Ectopic KRP1, KRP2 and KRP4 expression resulted in decreased gall size by inhibiting mitotic activity, whereas KRP6 induces mitosis in galls. Herein, we investigate the role of KRP3, KRP5 and KRP7 during gall development and compared their role with previously studied members of this class of cell cycle inhibitors. Overexpression of KRP3 and KRP7 culminated in undersized giant cells, with KRP3OE galls presenting peculiar elongated giant cells. Nuclei in KRP3OE and KRP5OE lines presented a convoluted and apparently connected phenotype. This appearance may be associated with the punctuated protein nuclear localization driven by specific common motifs. As well, ectopic expression of KRP3OE and KRP5OE affected nematode development and offspring. Decreased mitotic activity in galls of KRP3OE and KRP7OE lines led to a reduced gall size which presented distinct shapes - from more elongated like in the KRP3OE line to small rounded like in the KRP7OE line. Results presented strongly support the idea that induced expression of cell cycle inhibitors such as KRP3 and KRP7 in galls can be envisaged as a conceivable strategy for nematode feeding site control in crop species attacked by phytopathogenic nematodes.

Enriched environment decreases microglia and brain macrophages inflammatory phenotypes through adiponectin-dependent mechanisms: Relevance to depressive-like behavior.

Regulation of neuroinflammation by glial cells plays a major role in the pathophysiology of major depression. While astrocyte involvement has been well described, the role of microglia is still elusive. Recently, we have shown that Adiponectin (ApN) plays a crucial role in the anxiolytic/antidepressant neurogenesis-independent effects of enriched environment (EE) in mice; however its mechanisms of action within the brain remain unknown. Here, we show that in a murine model of depression induced by chronic corticosterone administration, the hippocampus and the hypothalamus display increased levels of inflammatory cytokines mRNA, which is reversed by EE housing. By combining flow cytometry, cell sorting and q-PCR, we show that microglia from depressive-like mice adopt a pro-inflammatory phenotype characterized by higher expression levels of IL-1ß, IL-6, TNF-α and IκB-α mRNAs. EE housing blocks pro-inflammatory cytokine gene induction and promotes arginase 1 mRNA expression in brain-sorted microglia, indicating that EE favors an anti-inflammatory activation state. We show that microglia and brain-macrophages from corticosterone-treated mice adopt differential expression profiles for CCR2, MHC class II and IL-4recα surface markers depending on whether the mice are kept in standard environment or EE. Interestingly, the effects of EE were abolished when cells are isolated from ApN knock-out mouse brains. When injected intra-cerebroventricularly, ApN, whose level is specifically increased in cerebrospinal fluid of depressive mice raised in EE, rescues microglia phenotype, reduces pro-inflammatory cytokine production by microglia and blocks depressive-like behavior in corticosterone-treated mice. Our data suggest that EE-induced ApN increase within the brain regulates microglia and brain macrophages phenotype and activation state, thus reducing neuroinflammation and depressive-like behaviors in mice.

Molecular and cellular neuroinflammatory status of mouse brain after systemic lipopolysaccharide challenge: importance of CCR2/CCL2 signaling.

BACKGROUND: Genetic and environmental factors are critical elements influencing the etiology of major depression. It is now accepted that neuroinflammatory processes play a major role in neuropsychological disorders. Neuroinflammation results from the dysregulation of the synthesis and/or release of pro- and anti-inflammatory cytokines with central or peripheral origin after various insults. Systemic bacterial lipopolysaccharide (LPS) challenge is commonly used to study inflammation-induced depressive-like behaviors in rodents. In the present study, we investigated immune-to-brain communication in mice by examining the effects of peripheral LPS injection on neuroinflammation encompassing cytokine and chemokine production, microglia and central nervous system (CNS)-associated phagocyte activation, immune cell infiltration and serotonergic neuronal function. METHODS: LPS was administered to C57BL/6 J mice by intraperitoneal injection; brains were collected and pro-inflammatory cytokine mRNA and proteins were measured. To examine the relative contribution of the different populations of brain immune cells to the occurrence of neuroinflammation after acute systemic inflammation, we precisely characterized them by flow cytometry, studied changes in their proportions and level of activation, and measured the amount of cytokines they released by Cytometric Bead Array™ after cell sorting and ex vivo culture. Because of the central role that the chemokine CCL2 seems to play in our paradigm, we studied the effect of CCL2 on the activity of serotonergic neurons of the raphe nucleus using electrophysiological recordings. RESULTS: We report that systemic LPS administration in mice caused a marked increase in pro-inflammatory IL-1ß, IL-6, TNFα and CCL2 (monocyte chemoattractant protein-1) mRNA and protein levels in the brain. Moreover, we found that LPS caused microglia and CNS-associated phagocyte activation characterized by upregulation of CCR2, TLR4/CD14, CD80 and IL-4Rα, associated with overproduction of pro-inflammatory cytokines and chemokines, especially CCL2. LPS also induced a marked and selective increase of CCR2(+) inflammatory monocytes within the brain. Finally, we showed that CCL2 hyperpolarized serotonergic raphe neurons in mouse midbrain slices, thus probably reducing the serotonin tone in projection areas. CONCLUSION: Together, we provide a detailed characterization of the molecular and cellular players involved in the establishment of neuroinflammation after systemic injection of LPS. This highlights the importance of the CCL2/CCR2 signaling and suggests a possible link with depressive disorders.

Memory CD8+ T cells mediate antibacterial immunity via CCL3 activation of TNF/ROI+ phagocytes.

Cytolysis, interferon gamma and tumor necrosis factor (TNF) alpha secretion are major effector mechanisms of memory CD8+ T cells that are believed to be required for immunological protection in vivo. By using mutants of the intracellular bacterium Listeria monocytogenes, we found that none of these effector activities is sufficient to protect against secondary infection with wild-type (WT) bacteria. We demonstrated that CCL3 derived from reactivated memory CD8+ T cells is required for efficient killing of WT bacteria. CCL3 induces a rapid TNF-alpha secretion by innate inflammatory mononuclear phagocytic cells (MPCs), which further promotes the production of radical oxygen intermediates (ROIs) by both MPCs and neutrophils. ROI generation is the final bactericidal mechanism involved in L. monocytogenes clearance. These results therefore uncover two levels of regulation of the antibacterial secondary protective response: (a) an antigen-dependent phase in which memory CD8+ T cells are reactivated and control the activation of the innate immune system, and (b) an antigen-independent phase in which the MPCs coordinate innate immunity and promote the bactericidal effector activities. In this context, CCL3-secreting memory CD8+ T cells are able to mediate "bystander" killing of an unrelated pathogen upon antigen-specific reactivation, a mechanism that may be important for the design of therapeutic vaccines.

Nanoscatterer-Assisted Fluorescence Amplification Technique.

Weak fluorescence signals, which are important in research and applications, are often masked by the background. Different amplification techniques are actively investigated. Here, a broadband, geometry-independent and flexible feedback scheme based on the random scattering of dielectric nanoparticles allows the amplification of a fluorescence signal by partial trapping of the radiation within the sample volume. Amplification of up to a factor of 40 is experimentally demonstrated in ultrapure water with dispersed TiO2 nanoparticles (30 to 50 nm in diameter) and fluorescein dye at 200 µmol concentration (pumped with 5 ns long, 3 mJ laser pulses at 490 nm). The measurements show a measurable reduction in linewidth at the emission peak, indicating that feedback-induced stimulated emission contributes to the large gain observed.

Natural killer cell behavior in lymph nodes revealed by static and real-time imaging.

Natural killer (NK) cells promote dendritic cell (DC) maturation and influence T cell differentiation in vitro. To better understand the nature of the putative interactions among these cells in vivo during the early phases of an adaptive immune response, we have used immunohistochemical analysis and dynamic intravital imaging to study NK cell localization and behavior in lymph nodes (LNs) in the steady state and shortly after infection with Leishmania major. In the LNs of naive mice, NK cells reside in the medulla and the paracortex, where they closely associate with DCs. In contrast to T cells, intravital microscopy revealed that NK cells in the superficial regions of LNs were slowly motile and maintained their interactions with DCs over extended times in the presence or absence of immune-activating signals. L. major induced NK cells to secrete interferon-gamma and to be recruited to the paracortex, where concomitant CD4 T cell activation occurred. Therefore, NK cells form a reactive but low mobile network in a strategic area of the LN where they can receive inflammatory signals, interact with DCs, and regulate colocalized T cell responses.

Splenic CD8α⁺ dendritic cells undergo rapid programming by cytosolic bacteria and inflammation to induce protective CD8⁺ T-cell memory.

Memory CD8(+) T lymphocytes are critical effector cells of the adaptive immune system mediating long-lived pathogen-specific protective immunity. Three signals - antigen, costimulation and inflammation - orchestrate optimal CD8(+) T-cell priming and differentiation into effector and memory cells and shape T-cell functional fate and ability to protect against challenge infections. While among the conventional spleen DCs (cDCs), the CD8α(+) but not the CD8α(-) cDCs most efficiently mediate CD8(+) T-cell priming, it is unclear which subset, irrespective of their capacity to process MHC class I-associated antigens, is most efficient at inducing naïve CD8(+) T-cell differentiation into pathogen-specific protective memory cells in vivo. Moreover, the origin of the required signals is still unclear. Using mice infected with the intracellular bacterium Listeria monocytogenes, we show that splenic CD8α(+) cDCs become endowed with all functional features to optimally prime protective memory CD8(+) T cells in vivo within only a few hours post-immunization. Such programming requires both cytosolic signals resulting from bacterial invasion of the host cells and extracellular inflammatory mediators. Thus, these data designate these cells as the best candidates to facilitate the development of cell-based vaccine therapy.

Direct visualization of peptide/MHC complexes at the surface and in the intracellular compartments of cells infected in vivo by Leishmania major.

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo.

Primed antigen-specific CD4+ T cells are required for NK cell activation in vivo upon Leishmania major infection.

The ability of NK cells to rapidly produce IFN-gamma is an important innate mechanism of resistance to many pathogens including Leishmania major. Molecular and cellular components involved in NK cell activation in vivo are still poorly defined, although a central role for dendritic cells has been described. In this study, we demonstrate that Ag-specific CD4(+) T cells are required to initiate NK cell activation early on in draining lymph nodes of L. major-infected mice. We show that early IFN-gamma secretion by NK cells is controlled by IL-2 and IL-12 and is dependent on CD40/CD40L interaction. These findings suggest that newly primed Ag-specific CD4(+) T cells could directly activate NK cells through the secretion of IL-2 but also indirectly through the regulation of IL-12 secretion by dendritic cells. Our results reveal an unappreciated role for Ag-specific CD4(+) T cells in the initiation of NK cell activation in vivo upon L. major infection and demonstrate bidirectional regulations between innate and adaptive immunity.

Redox-sensitive fluorescent biosensors detect Sinorhizobium meliloti intracellular redox changes under free-living and symbiotic lifestyles.

Reactive oxygen species such as hydrogen peroxide (H2O2) are key signaling molecules that control the setup and functioning of Rhizobium-legume symbiosis. This interaction results in the formation of a new organ, the root nodule, in which bacteria enter the host cells and differentiate into nitrogen (N2)-fixing bacteroids. The interaction between Sinorhizobium meliloti and Medicago truncatula is a genetic model to study N2-fixing symbiosis. In previous work, S. meliloti mutants impaired in the antioxidant defense, showed altered symbiotic properties, emphasizing the importance of redox-based regulation in the bacterial partner. However, direct measurements of S. meliloti intracellular redox state have never been performed. Here, we measured dynamic changes of intracellular H2O2 and glutathione redox potential by expressing roGFP2-Orp1 and Grx1-roGFP2 biosensors in S. meliloti. Kinetic analyses of redox changes under free-living conditions showed that these biosensors are suitable to monitor the bacterial redox state in real-time, after H2O2 challenge and in different genetic backgrounds. In planta, flow cytometry and confocal imaging experiments allowed the determination of sensor oxidation state in nodule bacteria. These cellular studies establish the existence of an oxidative shift in the redox status of S. meliloti during bacteroid differentiation. Our findings open up new possibilities for in vivo studies of redox dynamics during N2-fixing symbiosis.

Autofluorescence identifies highly phagocytic tissue-resident macrophages in mouse and human skin and cutaneous squamous cell carcinoma.

Macrophages from human and mouse skin share phenotypic and functional features, but remain to be characterized in pathological skin conditions. Skin-resident macrophages are known to derive from embryonic precursors or from adult hematopoiesis. In this report, we investigated the origins, phenotypes and functions of macrophage subsets in mouse and human skin and in cutaneous squamous cell carcinoma (cSCC) using the spectral flow cytometry technology that enables cell autofluorescence to be considered as a full-fledged parameter. Autofluorescence identifies macrophage subsets expressing the CD206 mannose receptor in human peri-tumoral skin and cSCC. In mouse, all AF+ macrophages express the CD206 marker, a subset of which also displaying the TIM-4 marker. While TIM-4-CD206+ AF+ macrophages can differentiate from bone-marrow monocytes and infiltrate skin and tumor, TIM-4 identifies exclusively a skin-resident AF+ macrophage subset that can derive from prenatal hematopoiesis which is absent in tumor core. In mouse and human, AF+ macrophages from perilesional skin and cSCC are highly phagocytic cells contrary to their AF- counterpart, thus identifying autofluorescence as a bona fide marker for phagocytosis. Our data bring to light autofluorescence as a functional marker characterizing subsets of phagocytic macrophages in skin and cSCC. Autofluorescence can thus be considered as an attractive marker of function of macrophage subsets in pathological context.

Cutaneous Squamous Cell Carcinoma Development Is Associated with a Temporal Infiltration of ILC1 and NK Cells with Immune Dysfunctions.

NK cells and tissue-resident innate lymphoid cells (ILCs) are innate effectors found in the skin. To investigate their temporal dynamics and specific functions throughout the development of cutaneous squamous cell carcinoma (cSCC), we combined transcriptomic and immunophenotyping analyses in mouse and human cSCCs. We identified an infiltration of NK cells and ILC1s as well as the presence of a few ILC3s. Adoptive transfer of NK cells in NK cell‒ and ILC-deficient Nfil3-/- mice revealed a role for NK cells in early control of cSCC. During tumor progression, we identified a population skewing with the infiltration of atypical ILC1 secreting inflammatory cytokines but reduced levels of IFN-γ at the papilloma stage. NK cells and ILC1s were functionally impaired, with reduced cytotoxicity and IFN-γ secretion associated with the downregulation of activating receptors. They also showed a high degree of heterogeneity in mouse and human cSCCs with the expression of several markers of exhaustion, including TIGIT on NK cells and PD-1 and TIM-3 on ILC1s. Our data show an enrichment in inflammatory ILC1 at the precancerous stage together with impaired antitumor functions in NK cells and ILC1 that could contribute to the development of cSCC and thus suggest that future immunotherapies should take both ILC populations into account.

CD4+ T cell polarization in mice is modulated by strain-specific major histocompatibility complex-independent differences within dendritic cells.

Resistance and susceptibility to Leishmania major in mice are determined by multiple genes and correlate with the preferential development of Th1 and Th2 responses, respectively. Here, we found that CD11b+ dendritic cells (DCs) prime parasite-specific CD4+ T cells in both susceptible BALB/c (H2-d) and resistant B10.D2 (H2-d) mice. However, BALB/c and B10.D2 DCs from L. major-infected mice differ in their ability to polarize naive T cells into Th1 or Th2 effector cells. This difference is cell-intrinsic, is not restricted to H2-d mice, and is observed with both parasite-specific and allospecific CD4+ T cells. Thus, strain-specific differences within CD11b+ DCs influence the ability of inbred mice to mount polarized CD4+ T cell responses.