RESUMEN

Metabolic dysfunction-associated steatotic liver disease (MASLD) prevalence is increasing in parallel with an obesity pandemic, calling for novel strategies for prevention and treatment. We defined a circulating proteome of human MASLD across ≈7000 proteins in ≈5000 individuals from diverse, at-risk populations across the metabolic health spectrum, demonstrating reproducible diagnostic performance and specifying both known and novel metabolic pathways relevant to MASLD (central carbon and amino acid metabolism, hepatocyte regeneration, inflammation, fibrosis, insulin sensitivity). A parsimonious proteomic signature of MASLD was associated with a protection from MASLD and its related multi-system metabolic consequences in >26000 free-living individuals, with an additive effect to polygenic risk. The MASLD proteome was encoded by genes that demonstrated transcriptional enrichment in liver, with spatial transcriptional activity in areas of steatosis in human liver biopsy and dynamicity for select targets in human liver across stages of steatosis. We replicated several top relations from proteomics and spatial tissue transcriptomics in a humanized "liver-on-a-chip" model of MASLD, highlighting the power of a full translational approach to discovery in MASLD. Collectively, these results underscore utility of blood-based proteomics as a dynamic "liquid biopsy" of human liver relevant to clinical biomarker and mechanistic applications.

RESUMEN

Improvements in COVID-19 treatments, especially for the critically ill, require deeper understanding of the mechanisms driving disease pathology. The complement system is not only a crucial component of innate host defense but can also contribute to tissue injury. Although all complement pathways have been implicated in COVID-19 pathogenesis, the upstream drivers and downstream effects on tissue injury remain poorly defined. We demonstrate that complement activation is primarily mediated by the alternative pathway, and we provide a comprehensive atlas of the complement alterations around the time of respiratory deterioration. Proteomic and single-cell sequencing mapping across cell types and tissues reveals a division of labor between lung epithelial, stromal, and myeloid cells in complement production, in addition to liver-derived factors. We identify IL-6 and STAT1/3 signaling as an upstream driver of complement responses, linking complement dysregulation to approved COVID-19 therapies. Furthermore, an exploratory proteomic study indicates that inhibition of complement C5 decreases epithelial damage and markers of disease severity. Collectively, these results support complement dysregulation as a key druggable feature of COVID-19.

Asunto(s)

COVID-19 , Interleucina-6 , Humanos , Proteómica , Proteínas del Sistema Complemento , Activación de Complemento

RESUMEN

Dendritic cells (DCs) mature in an immunogenic or tolerogenic manner depending on the context in which an antigen is perceived, preserving the balance between immunity and tolerance. Whereas the pathways driving immunogenic maturation in response to infectious insults are well-characterized, the signals that drive tolerogenic maturation during homeostasis are still poorly understood. We found that the engulfment of apoptotic cells triggered homeostatic maturation of type 1 conventional DCs (cDC1s) within the spleen. This maturation process could be mimicked by engulfment of empty, nonadjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and was highly specific to cDC1s. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to the activation of the liver X receptor (LXR) pathway, which promotes the efflux of cellular cholesterol, and repressed genes associated with immunogenic maturation. In contrast, simultaneous engagement of TLR3 to mimic viral infection via administration of poly(I:C)-adjuvanted LNPs repressed the LXR pathway, thus delaying cellular cholesterol efflux and inducing genes that promote T cell-mediated immunity. These data demonstrate that conserved cellular cholesterol efflux pathways are differentially regulated in tolerogenic versus immunogenic cDC1s and suggest that administration of nonadjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation.

Asunto(s)

Células Dendríticas , Transducción de Señal , Receptores X del Hígado/metabolismo , Transducción de Señal/genética , Homeostasis , Colesterol

RESUMEN

GM-CSF promotes myelopoiesis and inflammation, and GM-CSF blockade is being evaluated as a treatment for COVID-19-associated hyperinflammation. Alveolar GM-CSF is, however, required for monocytes to differentiate into alveolar macrophages (AMs) that control alveolar homeostasis. By mapping cross-species AM development to clinical lung samples, we discovered that COVID-19 is marked by defective GM-CSF-dependent AM instruction and accumulation of pro-inflammatory macrophages. In a multi-center, open-label RCT in 81 non-ventilated COVID-19 patients with respiratory failure, we found that inhalation of rhu-GM-CSF did not improve mean oxygenation parameters compared with standard treatment. However, more patients on GM-CSF had a clinical response, and GM-CSF inhalation induced higher numbers of virus-specific CD8 effector lymphocytes and class-switched B cells, without exacerbating systemic hyperinflammation. This translational proof-of-concept study provides a rationale for further testing of inhaled GM-CSF as a non-invasive treatment to improve alveolar gas exchange and simultaneously boost antiviral immunity in COVID-19. This study is registered at ClinicalTrials.gov (NCT04326920) and EudraCT (2020-001254-22).

Asunto(s)

COVID-19 , Macrófagos Alveolares , Humanos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Pulmón , Macrófagos

RESUMEN

Brain-immune cross-talk and neuroinflammation critically shape brain physiology in health and disease. A detailed understanding of the brain immune landscape is essential for developing new treatments for neurological disorders. Single-cell technologies offer an unbiased assessment of the heterogeneity, dynamics and functions of immune cells. Here we provide a protocol that outlines all the steps involved in performing single-cell multi-omic analysis of the brain immune compartment. This includes a step-by-step description on how to microdissect the border regions of the mouse brain, together with dissociation protocols tailored to each of these tissues. These combine a high yield with minimal dissociation-induced gene expression changes. Next, we outline the steps involved for high-dimensional flow cytometry and droplet-based single-cell RNA sequencing via the 10x Genomics platform, which can be combined with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and offers a higher throughput than plate-based methods. Importantly, we detail how to implement CITE-seq with large antibody panels to obtain unbiased protein-expression screening coupled to transcriptome analysis. Finally, we describe the main steps involved in the analysis and interpretation of the data. This optimized workflow allows for a detailed assessment of immune cell heterogeneity and activation in the whole brain or specific border regions, at RNA and protein level. The wet lab workflow can be completed by properly trained researchers (with basic proficiency in cell and molecular biology) and takes between 6 and 11 h, depending on the chosen procedures. The computational analysis requires a background in bioinformatics and programming in R.

Asunto(s)

Secuenciación de Nucleótidos de Alto Rendimiento , ARN , Animales , Encéfalo , Epítopos , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ratones , ARN/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcriptoma

RESUMEN

BACKGROUND: Nonclustered mouse protocadherin genes (Pcdh) encode proteins with a typical single ectodomain and a cytoplasmic domain with conserved motifs completely different from those of classic cadherins. Alternative splice isoforms differ in the size of these cytoplasmic domains. In view of the compelling evidence for gene silencing of protocadherins in human tumors, we started investigations on Pcdh functions in mouse cancer models. METHODS: For Pcdh10, we generated two mouse lines: one with floxed exon 1, leading to complete Pcdh10 ablation upon Cre action, and one with floxed exons 2 and 3, leading to ablation of only the long isoforms of Pcdh10. In a mouse medulloblastoma model, we used GFAP-Cre action to locally ablate Pcdh10 in combination with Trp53 and Rb1 ablation. From auricular tumors, that also arose, we obtained tumor-derived cell lines, which were analyzed for malignancy in vitro and in vivo. By lentiviral transduction, we re-expressed Pcdh10 cDNAs. RNA-Seq analyses were performed on these cell families. RESULTS: Surprisingly, not only medulloblastomas were generated in our model but also tumors of tagged auricles (pinnae). For both tumor types, ablation of either all or only long isoforms of Pcdh10 aggravated the disease. We argued that the perichondrial stem cell compartment is at the origin of the pinnal tumors. Immunohistochemical analysis of these tumors revealed different subtypes. We obtained several pinnal-tumor derived (PTD) cell lines and analyzed these for anchorage-independent growth, invasion into collagen matrices, tumorigenicity in athymic mice. Re-expression of either the short or a long isoform of Pcdh10 in two PTD lines counteracted malignancy in all assays. RNA-Seq analyses of these two PTD lines and their respective Pcdh10-rescued cell lines allowed to identify many interesting differentially expressed genes, which were largely different in the two cell families. CONCLUSIONS: A new mouse model was generated allowing for the first time to examine the remarkable tumor suppression activity of protocadherin-10 in vivo. Despite lacking several conserved motifs, the short isoform of Pcdh10 was fully active as tumor suppressor. Our model contributes to scrutinizing the complex molecular mechanisms of tumor initiation and progression upon PCDH10 silencing in many human cancers.

Asunto(s)

Neoplasias Cerebelosas , Meduloblastoma , Animales , Apoptosis/genética , Movimiento Celular/genética , Proliferación Celular/genética , Humanos , Meduloblastoma/genética , Ratones , Isoformas de Proteínas/genética , Protocadherinas

RESUMEN

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.

Asunto(s)

Evolución Biológica , Hepatocitos/metabolismo , Macrófagos/metabolismo , Proteogenómica , Animales , Núcleo Celular/metabolismo , Hígado Graso/genética , Hígado Graso/patología , Homeostasis , Humanos , Macrófagos del Hígado/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Lípidos/química , Hígado/metabolismo , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Células Mieloides/metabolismo , Obesidad/patología , Proteoma/metabolismo , Transducción de Señal , Transcriptoma/genética

RESUMEN

Macrophages are often prominently present in the tumor microenvironment, where distinct macrophage populations can differentially affect tumor progression. Although metabolism influences macrophage function, studies on the metabolic characteristics of ex vivo tumor-associated macrophage (TAM) subsets are rather limited. Using transcriptomic and metabolic analyses, we now reveal that pro-inflammatory major histocompatibility complex (MHC)-IIhi TAMs display a hampered tricarboxylic acid (TCA) cycle, while reparative MHC-IIlo TAMs show higher oxidative and glycolytic metabolism. Although both TAM subsets rapidly exchange lactate in high-lactate conditions, only MHC-IIlo TAMs use lactate as an additional carbon source. Accordingly, lactate supports the oxidative metabolism in MHC-IIlo TAMs, while it decreases the metabolic activity of MHC-IIhi TAMs. Lactate subtly affects the transcriptome of MHC-IIlo TAMs, increases L-arginine metabolism, and enhances the T cell suppressive capacity of these TAMs. Overall, our data uncover the metabolic intricacies of distinct TAM subsets and identify lactate as a carbon source and metabolic and functional regulator of TAMs.

Asunto(s)

Carcinoma Pulmonar de Lewis/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Lactatos/metabolismo , Neoplasias Pulmonares/patología , Linfocitos T/inmunología , Microambiente Tumoral , Macrófagos Asociados a Tumores/inmunología , Animales , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Femenino , Glucólisis , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Complejo Mayor de Histocompatibilidad , Metaboloma , Ratones , Ratones Endogámicos C57BL , Transcriptoma

RESUMEN

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.

Asunto(s)

Ácidos Grasos/metabolismo , Obesidad/patología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Tejido Adiposo Blanco/metabolismo , Animales , Citocinas/genética , Citocinas/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hidroliasas/genética , Hidroliasas/metabolismo , Resistencia a la Insulina , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Obesidad/metabolismo , Consumo de Oxígeno , Palmitatos/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/deficiencia , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Glioblastomas are aggressive primary brain cancers that recur as therapy-resistant tumors. Myeloid cells control glioblastoma malignancy, but their dynamics during disease progression remain poorly understood. Here, we employed single-cell RNA sequencing and CITE-seq to map the glioblastoma immune landscape in mouse tumors and in patients with newly diagnosed disease or recurrence. This revealed a large and diverse myeloid compartment, with dendritic cell and macrophage populations that were conserved across species and dynamic across disease stages. Tumor-associated macrophages (TAMs) consisted of microglia- or monocyte-derived populations, with both exhibiting additional heterogeneity, including subsets with conserved lipid and hypoxic signatures. Microglia- and monocyte-derived TAMs were self-renewing populations that competed for space and could be depleted via CSF1R blockade. Microglia-derived TAMs were predominant in newly diagnosed tumors, but were outnumbered by monocyte-derived TAMs following recurrence, especially in hypoxic tumor environments. Our results unravel the glioblastoma myeloid landscape and provide a framework for future therapeutic interventions.

Asunto(s)

Neoplasias Encefálicas/inmunología , Glioblastoma/inmunología , Macrófagos Asociados a Tumores/citología , Macrófagos Asociados a Tumores/inmunología , Animales , Humanos , Ratones , Análisis de la Célula Individual

RESUMEN

BACKGROUND: Modulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker. METHODS: We employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models. RESULTS: We were able to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs. CONCLUSIONS: Collectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy.

Asunto(s)

Antineoplásicos Inmunológicos/farmacología , Carcinoma Pulmonar de Lewis/terapia , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Depleción Linfocítica , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptores CCR8/deficiencia , Neoplasias Cutáneas/terapia , Linfocitos T Reguladores/inmunología , Animales , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/metabolismo , Terapia Combinada , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Fenotipo , Receptor de Muerte Celular Programada 1/metabolismo , RNA-Seq , Receptores CCR8/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Linfocitos T Reguladores/metabolismo

RESUMEN

Antigen-presenting conventional dendritic cells (cDCs) are broadly divided into type 1 and type 2 subsets that further adapt their phenotype and function to perform specialized tasks in the immune system. The precise signals controlling tissue-specific adaptation and differentiation of cDCs are currently poorly understood. We found that mice deficient in the Ste20 kinase Thousand and One Kinase 3 (TAOK3) lacked terminally differentiated ESAM+ CD4+ cDC2s in the spleen and failed to prime CD4+ T cells in response to allogeneic red-blood-cell transfusion. These NOTCH2- and ADAM10-dependent cDC2s were absent selectively in the spleen, but not in the intestine of Taok3-/- and CD11c-cre Taok3fl/fl mice. The loss of splenic ESAM+ cDC2s was cell-intrinsic and could be rescued by conditional overexpression of the constitutively active NOTCH intracellular domain in CD11c-expressing cells. Therefore, TAOK3 controls the terminal differentiation of NOTCH2-dependent splenic cDC2s.

Asunto(s)

Diferenciación Celular , Células Dendríticas/citología , Células Dendríticas/enzimología , Proteínas Quinasas/metabolismo , Receptor Notch2/metabolismo , Bazo/citología , Animales , Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/inmunología , Regulación de la Expresión Génica , Intestino Delgado/metabolismo , Ratones Endogámicos C57BL , Fenotipo , Dominios Proteicos , Proteínas Quinasas/deficiencia , Receptor Notch2/química , Transducción de Señal

RESUMEN

Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD.

Asunto(s)

Células de la Médula Ósea/citología , Activación de Macrófagos/inmunología , Macrófagos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Osteopontina/metabolismo , Animales , Biomarcadores/metabolismo , Células Cultivadas , Desmina/metabolismo , Femenino , Macrófagos del Hígado/citología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteoma/metabolismo , Transcriptoma/genética

RESUMEN

The phenotypic and functional dichotomy between IRF8+ type 1 and IRF4+ type 2 conventional dendritic cells (cDC1s and cDC2s, respectively) is well accepted; it is unknown how robust this dichotomy is under inflammatory conditions, when additionally monocyte-derived cells (MCs) become competent antigen-presenting cells (APCs). Using single-cell technologies in models of respiratory viral infection, we found that lung cDC2s acquired expression of the Fc receptor CD64 shared with MCs and of IRF8 shared with cDC1s. These inflammatory cDC2s (inf-cDC2s) were superior in inducing CD4+ T helper (Th) cell polarization while simultaneously presenting antigen to CD8+ T cells. When carefully separated from inf-cDC2s, MCs lacked APC function. Inf-cDC2s matured in response to cell-intrinsic Toll-like receptor and type 1 interferon receptor signaling, upregulated an IRF8-dependent maturation module, and acquired antigens via convalescent serum and Fc receptors. Because hybrid inf-cDC2s are easily confused with monocyte-derived cells, their existence could explain why APC functions have been attributed to MCs.

Asunto(s)

Plasticidad de la Célula/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunidad , Macrófagos/inmunología , Macrófagos/metabolismo , Infecciones por Respirovirus/etiología , Presentación de Antígeno , Biomarcadores , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Inmunofenotipificación , Interferón Tipo I/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Especificidad de Órganos/inmunología , Receptores Fc/metabolismo , Infecciones por Respirovirus/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factores de Transcripción , Virosis/genética , Virosis/inmunología , Virosis/metabolismo , Virosis/virología

RESUMEN

The increasing prevalence of obesity is accompanied by a rising incidence in metabolic syndrome and related pathologies such as non-alcoholic fatty liver disease. Macrophages are hypothesized to play central roles in these diseases, through their role as inflammatory mediators and as such are thought to be potential targets for future therapies. Recently, single cell technologies have revealed significant heterogeneity within the macrophage pool in both liver and adipose tissue in obesity. Thus current efforts are focused on dissecting this heterogeneity and understanding the distinct functions of the individual subsets. In this review, we discuss the current knowledge regarding macrophage heterogeneity, ontogeny and functions in the context of obese adipose tissue and fatty liver disease and attempt to align the distinct populations described to date.

Asunto(s)

Mediadores de Inflamación/inmunología , Macrófagos/inmunología , Síndrome Metabólico/patología , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/complicaciones , Animales , Humanos , Síndrome Metabólico/etiología , Enfermedad del Hígado Graso no Alcohólico/etiología

RESUMEN

While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: Relmα+Mgl1+ snMacs in the epineurium and Relmα-Mgl1- snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages.

Asunto(s)

Macrófagos/citología , Macrófagos/fisiología , Nervio Ciático/inmunología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Compresión Nerviosa , Transcriptoma

RESUMEN

Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation.

Asunto(s)

Trampas Extracelulares/metabolismo , Neutrófilos/metabolismo , Piel/patología , Proteína HMGB1/metabolismo , Humanos , Microambiente Tumoral , Cicatrización de Heridas

RESUMEN

Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity.

Asunto(s)

Células Endoteliales/fisiología , Células Estrelladas Hepáticas/fisiología , Hepatocitos/fisiología , Macrófagos del Hígado/fisiología , Hígado/citología , Macrófagos/fisiología , Monocitos/fisiología , Animales , Comunicación Celular , Diferenciación Celular , Células Cultivadas , Microambiente Celular , Femenino , Regulación de la Expresión Génica , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Receptores X del Hígado/genética , Receptores X del Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Notch/metabolismo

RESUMEN

Naive CD4+ T cells differentiate into functionally diverse T helper (Th) cell subsets. Th2 cells play a pathogenic role in asthma, yet a clear picture of their transcriptional profile is lacking. We performed single-cell RNA sequencing (scRNA-seq) of T helper cells from lymph node, lung, and airways in the house dust mite (HDM) model of allergic airway disease. scRNA-seq resolved transcriptional profiles of naive CD4+ T, Th1, Th2, regulatory T (Treg) cells, and a CD4+ T cell population responsive to type I interferons. Th2 cells in the airways were enriched for transcription of many genes, including Cd200r1, Il6, Plac8, and Igfbp7, and their mRNA profile was supported by analysis of chromatin accessibility and flow cytometry. Pathways associated with lipid metabolism were enriched in Th2 cells, and experiments with inhibitors of key metabolic pathways supported roles for glucose and lipid metabolism. These findings provide insight into the differentiation of pathogenic Th2 cells in the context of allergy.

Asunto(s)

Asma/inmunología , Hipersensibilidad Respiratoria/inmunología , Sistema Respiratorio/inmunología , Subgrupos de Linfocitos T/inmunología , Células Th2/inmunología , Animales , Antígenos Dermatofagoides/inmunología , Modelos Animales de Enfermedad , Humanos , Metabolismo de los Lípidos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Orexina/genética , Pyroglyphidae/inmunología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transcriptoma

RESUMEN

While the roles of parenchymal microglia in brain homeostasis and disease are fairly clear, other brain-resident myeloid cells remain less well understood. By dissecting border regions and combining single-cell RNA-sequencing with high-dimensional cytometry, bulk RNA-sequencing, fate-mapping and microscopy, we reveal the diversity of non-parenchymal brain macrophages. Border-associated macrophages (BAMs) residing in the dura mater, subdural meninges and choroid plexus consisted of distinct subsets with tissue-specific transcriptional signatures, and their cellular composition changed during postnatal development. BAMs exhibited a mixed ontogeny, and subsets displayed distinct self-renewal capacity following depletion and repopulation. Single-cell and fate-mapping analysis both suggested that there is a unique microglial subset residing on the apical surface of the choroid plexus epithelium. Finally, gene network analysis and conditional deletion revealed IRF8 as a master regulator that drives the maturation and diversity of brain macrophages. Our results provide a framework for understanding host-macrophage interactions in both the healthy and diseased brain.

Asunto(s)

Encéfalo/citología , Factores Reguladores del Interferón/metabolismo , Macrófagos/citología , Macrófagos/fisiología , Animales , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/citología