RESUMO
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.
Assuntos
Evolução Biológica , Hepatócitos/metabolismo , Macrófagos/metabolismo , Proteogenômica , Animais , Núcleo Celular/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Homeostase , Humanos , Células de Kupffer/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Lipídeos/química , Fígado/metabolismo , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Células Mieloides/metabolismo , Obesidade/patologia , Proteoma/metabolismo , Transdução de Sinais , Transcriptoma/genéticaRESUMO
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.
Assuntos
Plasticidade Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Imunidade , Macrófagos/imunologia , Macrófagos/metabolismo , Infecções por Respirovirus/etiologia , Apresentação de Antígeno , Biomarcadores , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Imunofenotipagem , Interferon Tipo I/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Especificidade de Órgãos/imunologia , Receptores Fc/metabolismo , Infecções por Respirovirus/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fatores de Transcrição , Viroses/genética , Viroses/imunologia , Viroses/metabolismo , Viroses/virologiaRESUMO
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.
Assuntos
Células da Medula Óssea/citologia , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Osteopontina/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Desmina/metabolismo , Feminino , Células de Kupffer/citologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteoma/metabolismo , Transcriptoma/genéticaRESUMO
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.
Assuntos
Asma/imunologia , Hipersensibilidade Respiratória/imunologia , Sistema Respiratório/imunologia , Subpopulações de Linfócitos T/imunologia , Células Th2/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Modelos Animais de Doenças , Humanos , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Orexina/genética , Pyroglyphidae/imunologia , Análise de Sequência de RNA , Análise de Célula Única , TranscriptomaRESUMO
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.
Assuntos
Células Endoteliais/fisiologia , Células Estreladas do Fígado/fisiologia , Hepatócitos/fisiologia , Células de Kupffer/fisiologia , Fígado/citologia , Macrófagos/fisiologia , Monócitos/fisiologia , Animais , Comunicação Celular , Diferenciação Celular , Células Cultivadas , Microambiente Celular , Feminino , Regulação da Expressão Gênica , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Notch/metabolismoRESUMO
Heterogeneity between different macrophage populations has become a defining feature of this lineage. However, the conserved factors defining macrophages remain largely unknown. The transcription factor ZEB2 is best described for its role in epithelial to mesenchymal transition; however, its role within the immune system is only now being elucidated. We show here that Zeb2 expression is a conserved feature of macrophages. Using Clec4f-cre, Itgax-cre, and Fcgr1-cre mice to target five different macrophage populations, we found that loss of ZEB2 resulted in macrophage disappearance from the tissues, coupled with their subsequent replenishment from bone-marrow precursors in open niches. Mechanistically, we found that ZEB2 functioned to maintain the tissue-specific identities of macrophages. In Kupffer cells, ZEB2 achieved this by regulating expression of the transcription factor LXRα, removal of which recapitulated the loss of Kupffer cell identity and disappearance. Thus, ZEB2 expression is required in macrophages to preserve their tissue-specific identities.
Assuntos
Células de Kupffer/citologia , Receptores X do Fígado/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Animais , Linhagem da Célula/imunologia , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Células de Kupffer/imunologia , Fígado/citologia , Receptores X do Fígado/metabolismo , Pulmão/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The role of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in homeostasis of the immune system is incompletely understood. Here we found that dendritic cells (DCs) constitutively activated the UPR sensor IRE-1α and its target, the transcription factor XBP-1, in the absence of ER stress. Loss of XBP-1 in CD11c+ cells led to defects in phenotype, ER homeostasis and antigen presentation by CD8α+ conventional DCs, yet the closely related CD11b+ DCs were unaffected. Whereas the dysregulated ER in XBP-1-deficient DCs resulted from loss of XBP-1 transcriptional activity, the phenotypic and functional defects resulted from regulated IRE-1α-dependent degradation (RIDD) of mRNAs, including those encoding CD18 integrins and components of the major histocompatibility complex (MHC) class I machinery. Thus, a precisely regulated feedback circuit involving IRE-1α and XBP-1 controls the homeostasis of CD8α+ conventional DCs.
Assuntos
Apresentação Cruzada/imunologia , Proteínas de Ligação a DNA/imunologia , Células Dendríticas/imunologia , Endorribonucleases/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Desdobramento de Proteína , Fatores de Transcrição/imunologia , Resposta a Proteínas não Dobradas/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/metabolismo , Retículo Endoplasmático/imunologia , Endorribonucleases/metabolismo , Retroalimentação Fisiológica/fisiologia , Homeostase/imunologia , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição de Fator Regulador X , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
The gut microbiota regulate susceptibility to multiple human diseases. The Nlrp6-ASC inflammasome is widely regarded as a hallmark host innate immune axis that shapes the gut microbiota composition. This notion stems from studies reporting dysbiosis in mice lacking these inflammasome components when compared with non-littermate wild-type animals. Here, we describe microbial analyses in inflammasome-deficient mice while minimizing non-genetic confounders using littermate-controlled Nlrp6-deficient mice and ex-germ-free littermate-controlled ASC-deficient mice that were all allowed to shape their gut microbiota naturally after birth. Careful microbial phylogenetic analyses of these cohorts failed to reveal regulation of the gut microbiota composition by the Nlrp6- and ASC-dependent inflammasomes. Our results obtained in two geographically separated animal facilities dismiss a generalizable impact of Nlrp6- and ASC-dependent inflammasomes on the composition of the commensal gut microbiota and highlight the necessity for littermate-controlled experimental design in assessing the influence of host immunity on gut microbial ecology.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Bactérias/genética , Colite/imunologia , Disbiose/imunologia , Microbioma Gastrointestinal/imunologia , Inflamassomos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proteínas Adaptadoras de Sinalização CARD , Células Cultivadas , Colite/induzido quimicamente , Colite/microbiologia , Disbiose/microbiologia , Feminino , Patrimônio Genético , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota , RNA Ribossômico 16S/análise , Receptores de Superfície Celular/genética , Dodecilsulfato de SódioRESUMO
Tissue-resident macrophages can derive from yolk sac macrophages (YS-Macs), fetal liver monocytes (FL-MOs), or adult bone-marrow monocytes (BM-MOs). The relative capacity of these precursors to colonize a niche, self-maintain, and perform tissue-specific functions is unknown. We simultaneously transferred traceable YS-Macs, FL-MOs, and BM-MOs into the empty alveolar macrophage (AM) niche of neonatal Csf2rb(-/-) mice. All subsets produced AMs, but in competition preferential outgrowth of FL-MOs was observed, correlating with their superior granulocyte macrophage-colony stimulating factor (GM-CSF) reactivity and proliferation capacity. When transferred separately, however, all precursors efficiently colonized the alveolar niche and generated AMs that were transcriptionally almost identical, self-maintained, and durably prevented alveolar proteinosis. Mature liver, peritoneal, or colon macrophages could not efficiently colonize the empty AM niche, whereas mature AMs could. Thus, precursor origin does not affect the development of functional self-maintaining tissue-resident macrophages and the plasticity of the mononuclear phagocyte system is largest at the precursor stage.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fígado/citologia , Macrófagos Alveolares/citologia , Saco Vitelino/citologia , Animais , Proliferação de Células , Subunidade beta Comum dos Receptores de Citocinas/genética , Fígado/embriologia , Fígado/imunologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transcriptoma/imunologia , Saco Vitelino/imunologiaRESUMO
Interferon regulatory factor-8 (IRF8) has been proposed to be essential for development of monocytes, plasmacytoid dendritic cells (pDCs) and type 1 conventional dendritic cells (cDC1s) and remains highly expressed in differentiated DCs. Transcription factors that are required to maintain the identity of terminally differentiated cells are designated "terminal selectors." Using BM chimeras, conditional Irf8(fl/fl) mice and various promotors to target Cre recombinase to different stages of monocyte and DC development, we have identified IRF8 as a terminal selector of the cDC1 lineage controlling survival. In monocytes, IRF8 was necessary during early but not late development. Complete or late deletion of IRF8 had no effect on pDC development or survival but altered their phenotype and gene-expression profile leading to increased T cell stimulatory function but decreased type 1 interferon production. Thus, IRF8 differentially controls the survival and function of terminally differentiated monocytes, cDC1s, and pDCs.
Assuntos
Diferenciação Celular/fisiologia , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Fatores Reguladores de Interferon/metabolismo , Fatores de Transcrição/metabolismo , Animais , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Monócitos/fisiologia , Regiões Promotoras Genéticas/fisiologia , Linfócitos T/metabolismo , Linfócitos T/fisiologiaRESUMO
Targeted mutagenesis in mice is a powerful tool for functional analysis of genes. However, genetic variation between embryonic stem cells (ESCs) used for targeting (previously almost exclusively 129-derived) and recipient strains (often C57BL/6J) typically results in congenic mice in which the targeted gene is flanked by ESC-derived passenger DNA potentially containing mutations. Comparative genomic analysis of 129 and C57BL/6J mouse strains revealed indels and single nucleotide polymorphisms resulting in alternative or aberrant amino acid sequences in 1,084 genes in the 129-strain genome. Annotating these passenger mutations to the reported genetically modified congenic mice that were generated using 129-strain ESCs revealed that nearly all these mice possess multiple passenger mutations potentially influencing the phenotypic outcome. We illustrated this phenotypic interference of 129-derived passenger mutations with several case studies and developed a Me-PaMuFind-It web tool to estimate the number and possible effect of passenger mutations in transgenic mice of interest.
Assuntos
Variação Genética/genética , Genoma/genética , Camundongos Endogâmicos C57BL/genética , Sequência de Aminoácidos/genética , Animais , Caspases/genética , Caspases Iniciadoras , Mapeamento Cromossômico , Hibridização Genômica Comparativa , Conexinas/genética , Genótipo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 8 da Matriz/genética , Camundongos , Camundongos Congênicos/genética , Camundongos Knockout , Mutação/genética , Proteínas do Tecido Nervoso/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
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.
Assuntos
Diferenciação Celular , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Proteínas Quinases/metabolismo , Receptor Notch2/metabolismo , Baço/citologia , Animais , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica , Intestino Delgado/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo , Domínios Proteicos , Proteínas Quinases/deficiência , Receptor Notch2/química , Transdução de SinaisRESUMO
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.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Animais , Apoptose/genética , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Meduloblastoma/genética , Camundongos , Isoformas de Proteínas/genética , ProtocaderinasRESUMO
Norovirus infection is the leading cause of food-borne gastroenteritis worldwide, being responsible for over 200,000 deaths annually. Studies with murine norovirus (MNV) showed that protective STAT1 signaling controls viral replication and pathogenesis, but the immune mechanisms that noroviruses exploit to induce pathology are elusive. Here, we show that gastrointestinal MNV infection leads to widespread IL-1ß maturation in MNV-susceptible STAT1-deficient mice. MNV activates the canonical Nlrp3 inflammasome in macrophages, leading to maturation of IL-1ß and to Gasdermin D (GSDMD)-dependent pyroptosis. STAT1-deficient macrophages displayed increased MAVS-mediated expression of pro-IL-1ß, facilitating elevated Nlrp3-dependent release of mature IL-1ß upon MNV infection. Accordingly, MNV-infected Stat1-/- mice showed Nlrp3-dependent maturation of IL-1ß as well as Nlrp3-dependent pyroptosis as assessed by in vivo cleavage of GSDMD to its active N-terminal fragment. While MNV-induced diarrheic responses were not affected, Stat1-/- mice additionally lacking either Nlrp3 or GSDMD displayed lower levels of the fecal inflammatory marker Lipocalin-2 as well as delayed lethality after gastrointestinal MNV infection. Together, these results uncover new insights into the mechanisms of norovirus-induced inflammation and cell death, thereby revealing Nlrp3 inflammasome activation and ensuing GSDMD-driven pyroptosis as contributors to MNV-induced immunopathology in susceptible STAT1-deficient mice.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Infecções por Caliciviridae/patologia , Trato Gastrointestinal/patologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/fisiologia , Fator de Transcrição STAT1/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/virologia , Células Cultivadas , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/virologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Norovirus/imunologia , Norovirus/patogenicidade , Proteínas de Ligação a FosfatoRESUMO
In mammals, macrophages (MF) are present in virtually all tissues where they serve many different functions linked primarily to the maintenance of homeostasis, innate defense against pathogens, tissue repair and metabolism. Although some of these functions appear common to all tissues, others are specific to the homing tissue. Thus, MF become adapted to perform particular functions in a given tissue. Accordingly, MF express common markers but also sets of tissue-specific markers linked to dedicated functions. One of the largest pool of MF in the body lines up the wall of the gut. Located in the small intestine, Peyer's patches (PP) are primary antigen sampling and mucosal immune response inductive sites. Surprisingly, although markers of intestinal MF, such as F4/80, have been identified more than 30â¯years ago, MF of PP escaped any kind of phenotypic description and remained "unknown" for decades. In absence of MF identification, the characterization of the PP mononuclear phagocyte system (MPS) functions has been impaired. However, taking into account that PP are privileged sites of entry for pathogens, it is important to understand how the latter are handled by and/or escape the PP MPS, especially MF, which role in killing invaders is well known. This review focuses on recent advances on the PP MPS, which have allowed, through new criteria of PP phagocyte subset identification, the characterization of PP MF origin, diversity, specificity, location and functions.
Assuntos
Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Macrófagos/imunologia , Sistema Fagocitário Mononuclear/imunologia , Nódulos Linfáticos Agregados/imunologia , Imunidade Adaptativa/imunologia , Animais , Imunidade nas Mucosas/imunologia , Sistema Fagocitário Mononuclear/citologia , Nódulos Linfáticos Agregados/citologia , Fagócitos/imunologiaRESUMO
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.
RESUMO
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.
Assuntos
COVID-19 , Interleucina-6 , Humanos , Proteômica , Proteínas do Sistema Complemento , Ativação do ComplementoRESUMO
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.
Assuntos
Células Dendríticas , Transdução de Sinais , Receptores X do Fígado/metabolismo , Transdução de Sinais/genética , Homeostase , ColesterolRESUMO
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.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , RNA , Animais , Encéfalo , Epitopos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , TranscriptomaRESUMO
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).