RESUMEN
Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.
Asunto(s)
Supervivencia Celular , Neuronas Dopaminérgicas , FN-kappa B , Factor de Necrosis Tumoral alfa , Proteína p53 Supresora de Tumor , Neuronas Dopaminérgicas/metabolismo , Animales , Humanos , FN-kappa B/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ratones , Supervivencia Celular/efectos de los fármacos , Transducción de Señal , Enfermedad de Parkinson/metabolismo , Células Madre Pluripotentes/metabolismo , Apoptosis , Modelos Animales de Enfermedad , Sistemas CRISPR-CasRESUMEN
Sepsis is a systemic response to infection with life-threatening consequences. Our understanding of the molecular and cellular impact of sepsis across organs remains rudimentary. Here, we characterize the pathogenesis of sepsis by measuring dynamic changes in gene expression across organs. To pinpoint molecules controlling organ states in sepsis, we compare the effects of sepsis on organ gene expression to those of 6 singles and 15 pairs of recombinant cytokines. Strikingly, we find that the pairwise effects of tumor necrosis factor plus interleukin (IL)-18, interferon-gamma or IL-1ß suffice to mirror the impact of sepsis across tissues. Mechanistically, we map the cellular effects of sepsis and cytokines by computing changes in the abundance of 195 cell types across 9 organs, which we validate by whole-mouse spatial profiling. Our work decodes the cytokine cacophony in sepsis into a pairwise cytokine message capturing the gene, cell and tissue responses of the host to the disease.
Asunto(s)
Citocinas , Sepsis , Ratones , Animales , Interleucina-6/genética , Factor de Necrosis Tumoral alfa/metabolismo , Interferón gamma , Sepsis/genéticaRESUMEN
Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.
Asunto(s)
Infecciones por Coronavirus/inmunología , Centro Germinal/inmunología , Neumonía Viral/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Anciano , Anciano de 80 o más Años , Linfocitos B/inmunología , COVID-19 , Femenino , Centro Germinal/patología , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Bazo/inmunología , Bazo/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cell function and activity are regulated through integration of signaling, epigenetic, transcriptional, and metabolic pathways. Here, we introduce INs-seq, an integrated technology for massively parallel recording of single-cell RNA sequencing (scRNA-seq) and intracellular protein activity. We demonstrate the broad utility of INs-seq for discovering new immune subsets by profiling different intracellular signatures of immune signaling, transcription factor combinations, and metabolic activity. Comprehensive mapping of Arginase 1-expressing cells within tumor models, a metabolic immune signature of suppressive activity, discovers novel Arg1+ Trem2+ regulatory myeloid (Mreg) cells and identifies markers, metabolic activity, and pathways associated with these cells. Genetic ablation of Trem2 in mice inhibits accumulation of intra-tumoral Mreg cells, leading to a marked decrease in dysfunctional CD8+ T cells and reduced tumor growth. This study establishes INs-seq as a broadly applicable technology for elucidating integrated transcriptional and intra-cellular maps and identifies the molecular signature of myeloid suppressive cells in tumors.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Neoplasias/patología , ARN Citoplasmático Pequeño/química , Receptores Inmunológicos/metabolismo , Animales , Arginasa/genética , Arginasa/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/metabolismo , ARN Citoplasmático Pequeño/metabolismo , Receptores Inmunológicos/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Microambiente Tumoral , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Cutaneous TRPV1+ neurons directly sense noxious stimuli, inflammatory cytokines, and pathogen-associated molecules and are required for innate immunity against some skin pathogens. Important unanswered questions are whether TRPV1+ neuron activation in isolation is sufficient to initiate innate immune responses and what is the biological function for TRPV1+ neuron-initiated immune responses. We used TRPV1-Ai32 optogenetic mice and cutaneous light stimulation to activate cutaneous neurons in the absence of tissue damage or pathogen-associated products. We found that TRPV1+ neuron activation was sufficient to elicit a local type 17 immune response that augmented host defense to C. albicans and S. aureus. Moreover, local neuron activation elicited type 17 responses and augmented host defense at adjacent, unstimulated skin through a nerve reflex arc. These data show the sufficiency of TRPV1+ neuron activation for host defense and demonstrate the existence of functional anticipatory innate immunity at sites adjacent to infection that depends on antidromic neuron activation.
Asunto(s)
Inmunidad Innata/inmunología , Interleucina-23/metabolismo , Interleucina-6/metabolismo , Células Receptoras Sensoriales/inmunología , Piel/inmunología , Canales Catiónicos TRPV/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Candida albicans/inmunología , Inflamación/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Optogenética/métodos , Piel/microbiología , Staphylococcus aureus/inmunología , Canales Catiónicos TRPV/genéticaRESUMEN
Necrosis of infected macrophages constitutes a critical pathogenetic event in tuberculosis by releasing mycobacteria into the growth-permissive extracellular environment. In zebrafish infected with Mycobacterium marinum or Mycobacterium tuberculosis, excess tumor necrosis factor triggers programmed necrosis of infected macrophages through the production of mitochondrial reactive oxygen species (ROS) and the participation of cyclophilin D, a component of the mitochondrial permeability transition pore. Here, we show that this necrosis pathway is not mitochondrion-intrinsic but results from an inter-organellar circuit initiating and culminating in the mitochondrion. Mitochondrial ROS induce production of lysosomal ceramide that ultimately activates the cytosolic protein BAX. BAX promotes calcium flow from the endoplasmic reticulum into the mitochondrion through ryanodine receptors, and the resultant mitochondrial calcium overload triggers cyclophilin-D-mediated necrosis. We identify ryanodine receptors and plasma membrane L-type calcium channels as druggable targets to intercept mitochondrial calcium overload and necrosis of mycobacterium-infected zebrafish and human macrophages.
Asunto(s)
Macrófagos/microbiología , Macrófagos/patología , Mitocondrias/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Tuberculosis/inmunología , Tuberculosis/patología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis , Calcio/metabolismo , Retículo Endoplásmico/microbiología , Humanos , Lisosomas/microbiología , Potencial de la Membrana Mitocondrial , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium marinum , Mycobacterium tuberculosis , Necrosis , Especies Reactivas de Oxígeno/metabolismo , Células THP-1 , Pez CebraRESUMEN
Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.
Asunto(s)
Colitis Ulcerosa/patología , Colon/metabolismo , Adulto , Anciano , Anticuerpos Monoclonales/uso terapéutico , Bestrofinas/metabolismo , Antígenos CD8/metabolismo , Estudios de Casos y Controles , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/metabolismo , Colon/patología , Enterocitos/citología , Enterocitos/metabolismo , Femenino , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Humanos , Interleucina-17/metabolismo , Masculino , Persona de Mediana Edad , Factores de Riesgo , Linfocitos T/citología , Linfocitos T/metabolismo , Trombospondinas/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adulto JovenRESUMEN
New opportunities are needed to increase immune checkpoint blockade (ICB) benefit. Whereas the interferon (IFN)γ pathway harbors both ICB resistance factors and therapeutic opportunities, this has not been systematically investigated for IFNγ-independent signaling routes. A genome-wide CRISPR/Cas9 screen to sensitize IFNγ receptor-deficient tumor cells to CD8 T cell elimination uncovered several hits mapping to the tumor necrosis factor (TNF) pathway. Clinically, we show that TNF antitumor activity is only limited in tumors at baseline and in ICB non-responders, correlating with its low abundance. Taking advantage of the genetic screen, we demonstrate that ablation of the top hit, TRAF2, lowers the TNF cytotoxicity threshold in tumors by redirecting TNF signaling to favor RIPK1-dependent apoptosis. TRAF2 loss greatly enhanced the therapeutic potential of pharmacologic inhibition of its interaction partner cIAP, another screen hit, thereby cooperating with ICB. Our results suggest that selective reduction of the TNF cytotoxicity threshold increases the susceptibility of tumors to immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunoterapia , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Interferón gamma/metabolismo , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias/mortalidad , Neoplasias/terapia , ARN Guía de Kinetoplastida/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Transducción de Señal/efectos de los fármacos , Factor 2 Asociado a Receptor de TNF/deficiencia , Factor 2 Asociado a Receptor de TNF/genética , Factor de Necrosis Tumoral alfa/farmacología , Receptor de Interferón gammaRESUMEN
Severe acute respiratory syndrome coronavirus 2 infections can cause coronavirus disease 2019 (COVID-19), which manifests with a range of severities from mild illness to life-threatening pneumonia and multi-organ failure. Severe COVID-19 is characterized by an inflammatory signature, including high levels of inflammatory cytokines, alveolar inflammatory infiltrates and vascular microthrombi. Here we show that patients with severe COVID-19 produced a unique serologic signature, including an increased likelihood of IgG1 with afucosylated Fc glycans. This Fc modification on severe acute respiratory syndrome coronavirus 2 IgGs enhanced interactions with the activating Fcγ receptor FcγRIIIa; when incorporated into immune complexes, Fc afucosylation enhanced production of inflammatory cytokines by monocytes, including interleukin-6 and tumor necrosis factor. These results show that disease severity in COVID-19 correlates with the presence of proinflammatory IgG Fc structures, including afucosylated IgG1.
Asunto(s)
COVID-19/inmunología , Citocinas/inmunología , Inmunoglobulina G/inmunología , Receptores de IgG/inmunología , SARS-CoV-2/inmunología , Adolescente , Adulto , Anciano , COVID-19/metabolismo , COVID-19/virología , Niño , Citocinas/metabolismo , Femenino , Glicosilación , Humanos , Inmunoglobulina G/metabolismo , Interleucina-6 , Masculino , Persona de Mediana Edad , Receptores de IgG/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Índice de Severidad de la Enfermedad , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRß signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.
Asunto(s)
Neoplasias Encefálicas/inmunología , Puntos de Control del Ciclo Celular , Glioblastoma/inmunología , Células Asesinas Naturales/inmunología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Neoplasias Encefálicas/patología , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Femenino , Glioblastoma/patología , Humanos , Inmunidad Innata , Interferón gamma/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor 2 Gatillante de la Citotoxidad Natural/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN-I). What other functions pDCs exert in vivo during viral infections is controversial, and more studies are needed to understand their orchestration. In the present study, we characterize in depth and link pDC activation states in animals infected by mouse cytomegalovirus by combining Ifnb1 reporter mice with flow cytometry, single-cell RNA sequencing, confocal microscopy and a cognate CD4 T cell activation assay. We show that IFN-I production and T cell activation were performed by the same pDC, but these occurred sequentially in time and in different micro-anatomical locations. In addition, we show that pDC commitment to IFN-I production was marked early on by their downregulation of leukemia inhibitory factor receptor and was promoted by cell-intrinsic tumor necrosis factor signaling. We propose a new model for how individual pDCs are endowed to exert different functions in vivo during a viral infection, in a manner tightly orchestrated in time and space.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Infecciones por Herpesviridae/inmunología , Muromegalovirus/fisiología , Animales , Células Cultivadas , Interferón Tipo I/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Análisis de Secuencia de ARN , Transducción de Señal , Análisis de la Célula Individual , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A20 is an anti-inflammatory protein that is strongly linked to human disease. Here, we find that mice expressing three distinct targeted mutations of A20's zinc finger 7 (ZF7) ubiquitin-binding motif uniformly developed digit arthritis with features common to psoriatic arthritis, while mice expressing point mutations in A20's OTU or ZF4 motifs did not exhibit this phenotype. Arthritis in A20ZF7 mice required T cells and MyD88, was exquisitely sensitive to tumor necrosis factor and interleukin-17A, and persisted in germ-free conditions. A20ZF7 cells exhibited prolonged IκB kinase activity that drove exaggerated transcription of late-phase nuclear factor-κB response genes in vitro and in prediseased mouse paws in vivo. In addition, mice expressing double-mutant A20 proteins in A20's ZF4 and ZF7 motifs died perinatally with multi-organ inflammation. Therefore, A20's ZF4 and ZF7 motifs synergistically prevent inflammatory disease in a non-catalytic manner.
Asunto(s)
Artritis Psoriásica/metabolismo , Inflamación/metabolismo , Ubiquitina/metabolismo , Animales , Células Cultivadas , Interleucina-17 , Ratones , Ratones Endogámicos C57BL , Mutación/genética , FN-kappa B/metabolismo , Unión Proteica/fisiología , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinación/fisiología , Dedos de Zinc/fisiologíaRESUMEN
Protein ubiquitination regulates protein stability and modulates the composition of signaling complexes. A20 is a negative regulator of inflammatory signaling, but the molecular mechanisms involved are ill understood. Here, we generated Tnfaip3 gene-targeted A20 mutant mice bearing inactivating mutations in the zinc finger 7 (ZnF7) and ZnF4 ubiquitin-binding domains, revealing that binding to polyubiquitin is essential for A20 to suppress inflammatory disease. We demonstrate that a functional ZnF7 domain was required for recruiting A20 to the tumor necrosis factor receptor 1 (TNFR1) signaling complex and to suppress inflammatory signaling and cell death. The combined inactivation of ZnF4 and ZnF7 phenocopied the postnatal lethality and severe multiorgan inflammation of A20-deficient mice. Conditional tissue-specific expression of mutant A20 further revealed the key role of ubiquitin-binding in myeloid and intestinal epithelial cells. Collectively, these results demonstrate that the anti-inflammatory and cytoprotective functions of A20 are largely dependent on its ubiquitin-binding properties.
Asunto(s)
Inflamación/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Epiteliales/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Poliubiquitina/metabolismo , Unión Proteica/fisiología , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Dedos de Zinc/fisiologíaRESUMEN
The cytokine interleukin (IL)-1ß is a key mediator of antimicrobial immunity as well as autoimmune inflammation. Production of IL-1ß requires transcription by innate immune receptor signaling and maturational cleavage by inflammasomes. Whether this mechanism applies to IL-1ß production seen in T cell-driven autoimmune diseases remains unclear. Here, we describe an inflammasome-independent pathway of IL-1ß production that was triggered upon cognate interactions between effector CD4+ T cells and mononuclear phagocytes (MPs). The cytokine TNF produced by activated CD4+ T cells engaged its receptor TNFR on MPs, leading to pro-IL-1ß synthesis. Membrane-bound FasL, expressed by CD4+ T cells, activated death receptor Fas signaling in MPs, resulting in caspase-8-dependent pro-IL-1ß cleavage. The T cell-instructed IL-1ß resulted in systemic inflammation, whereas absence of TNFR or Fas signaling protected mice from CD4+ T cell-driven autoimmunity. The TNFR-Fas-caspase-8-dependent pathway provides a mechanistic explanation for IL-1ß production and its consequences in CD4+ T cell-driven autoimmune pathology.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Inflamación/patología , Interleucina-1beta/metabolismo , Células Mieloides/metabolismo , Animales , Caspasa 1/genética , Caspasa 8/metabolismo , Células Cultivadas , Células Dendríticas/inmunología , Proteína Ligando Fas/metabolismo , Inmunidad Innata/inmunología , Inflamasomas/inmunología , Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium tuberculosis/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.
Asunto(s)
Apoptosis/inmunología , Caspasa 8/inmunología , Neutrófilos/inmunología , Proteínas Quinasas/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Animales , Caspasa 8/genética , Células Cultivadas , Eliminación de Gen , Inflamación/inmunología , Interleucina-1/inmunología , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Receptores Tipo I de Interleucina-1/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
A disintegrin and metalloprotease 17 (ADAM17) is a membrane-tethered protease that triggers multiple signaling pathways. It releases active forms of the primary inflammatory cytokine tumor necrosis factor (TNF) and cancer-implicated epidermal growth factor (EGF) family growth factors. iRhom2, a rhomboid-like, membrane-embedded pseudoprotease, is an essential cofactor of ADAM17. Here, we present cryoelectron microscopy (cryo-EM) structures of the human ADAM17/iRhom2 complex in both inactive and active states. These reveal three regulatory mechanisms. First, exploiting the rhomboid-like hallmark of TMD recognition, iRhom2 interacts with the ADAM17 TMD to promote ADAM17 trafficking and enzyme maturation. Second, a unique iRhom2 extracellular domain unexpectedly retains the cleaved ADAM17 inhibitory prodomain, safeguarding against premature activation and dysregulated proteolysis. Finally, loss of the prodomain from the complex mobilizes the ADAM17 protease domain, contributing to its ability to engage substrates. Our results reveal how a rhomboid-like pseudoprotease has been repurposed during evolution to regulate a potent membrane-tethered enzyme, ADAM17, ensuring the fidelity of inflammatory and growth factor signaling.
Asunto(s)
Proteína ADAM17 , Microscopía por Crioelectrón , Transducción de Señal , Proteína ADAM17/metabolismo , Proteína ADAM17/genética , Humanos , Células HEK293 , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Inflamación/metabolismo , Inflamación/genética , Proteolisis , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genética , Dominios Proteicos , Unión Proteica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/genética , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.
Asunto(s)
Enfermedad de Crohn/inmunología , Células Endoteliales/inmunología , Íleon/inmunología , Linfa/metabolismo , Vasos Linfáticos/inmunología , Mesenterio/inmunología , Estructuras Linfoides Terciarias/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ileítis , Linfangitis , Ratones , Ratones Noqueados , Estrés MecánicoRESUMEN
Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.
Asunto(s)
COVID-19/inmunología , Interferón-alfa/inmunología , Células Asesinas Naturales/inmunología , SARS-CoV-2/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Bases , Humanos , Inmunidad Innata/inmunología , Inflamación/inmunología , Interferón-alfa/sangre , Fibrosis Pulmonar/patología , RNA-Seq , Índice de Severidad de la Enfermedad , Transcriptoma/genética , Reino Unido , Estados UnidosRESUMEN
Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.
Asunto(s)
Vasos Sanguíneos/inmunología , Dermatitis por Contacto/inmunología , Inflamación/inmunología , Mastocitos/inmunología , Neutrófilos/inmunología , Piel/patología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Circulación Sanguínea , Degranulación de la Célula , Células Cultivadas , Enfermedades del Sistema Inmune , Trastornos Leucocíticos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Activación Neutrófila , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Vesículas Secretoras/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Lymphoid stromal cells (LSCs) are essential organizers of immune responses. We analyzed tonsillar tissue by combining flow cytometry, in situ imaging, RNA sequencing, and functional assays, defining three distinct human LSC subsets. The integrin CD49a designated perivascular stromal cells exhibiting features of local committed LSC precursors and segregated cytokine and chemokine-producing fibroblastic reticular cells (FRCs) supporting B and T cell survival. The follicular dendritic cell transcriptional profile reflected active responses to B cell and non-B cell stimuli. We therefore examined the effect of B cell stimuli on LSCs in follicular lymphoma (FL). FL B cells interacted primarily with CD49a+ FRCs. Transcriptional analyses revealed LSC reprogramming in situ downstream of the cytokines tumor necrosis factor (TNF) and transforming growth factor ß (TGF-ß), including increased expression of the chemokines CCL19 and CCL21. Our findings define human LSC populations in healthy tissue and reveal bidirectional crosstalk between LSCs and malignant B cells that may present a targetable axis in lymphoma.