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1.
Nat Immunol ; 23(4): 568-580, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35314846

RESUMEN

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.


Asunto(s)
Aminoaciltransferasas , Linfocitos T CD8-positivos , Quimiocinas , Neoplasias , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Animales , Linfocitos T CD8-positivos/patología , Quimiocinas/metabolismo , Inmunoterapia , Infiltración Leucémica , Ratones , Ratones Noqueados , Monocitos , Neoplasias/inmunología
2.
Nat Immunol ; 22(5): 571-585, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33903764

RESUMEN

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.


Asunto(s)
Células Dendríticas Foliculares/inmunología , Fibroblastos/inmunología , Ganglios Linfáticos/inmunología , Células del Estroma/inmunología , Anciano , Animales , Apoptosis/genética , Apoptosis/inmunología , Proliferación Celular/genética , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Dendríticas Foliculares/metabolismo , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica/inmunología , Técnicas de Sustitución del Gen , Humanos , Inmunidad Celular/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ganglios Linfáticos/citología , Masculino , Ratones , Ratones Transgénicos , RNA-Seq , Análisis de la Célula Individual , Células del Estroma/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo
3.
Immunity ; 54(7): 1511-1526.e8, 2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34260887

RESUMEN

Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.


Asunto(s)
Homeostasis/fisiología , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Monocitos/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Apoptosis/fisiología , Médula Ósea/metabolismo , Médula Ósea/patología , Células COS , Diferenciación Celular/fisiología , Línea Celular , Línea Celular Tumoral , Linaje de la Célula/fisiología , Proliferación Celular/fisiología , Chlorocebus aethiops , Femenino , Humanos , Pulmón/patología , Macrófagos Alveolares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/patología , Células Mieloides/metabolismo , Células Mieloides/patología , Metástasis de la Neoplasia/patología , Proteómica/métodos , Transducción de Señal/fisiología
4.
Immunity ; 52(2): 357-373.e9, 2020 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-32049051

RESUMEN

Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.


Asunto(s)
Macrófagos/inmunología , Proteínas de la Membrana/metabolismo , Neoplasias/inmunología , Nucleótidos Cíclicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Tirosina Quinasa c-Mer/inmunología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Antígeno B7-H1/inmunología , Células Cultivadas , Femenino , Inmunidad Innata , Inmunoterapia , Interferón Tipo I/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/terapia , Nucleotidiltransferasas/deficiencia , Nucleotidiltransferasas/metabolismo , Fagocitosis , Receptor de Muerte Celular Programada 1/inmunología , Receptores Purinérgicos P2X7/deficiencia , Transducción de Señal/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa c-Mer/genética
5.
Nature ; 611(7934): 148-154, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36171287

RESUMEN

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1-3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFß receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFß-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.


Asunto(s)
Fibroblastos Asociados al Cáncer , Proteínas de la Membrana , Miofibroblastos , Neoplasias Pancreáticas , Células del Estroma , Animales , Humanos , Ratones , Fibroblastos Asociados al Cáncer/metabolismo , Linfocitos T CD8-positivos/inmunología , Proteínas de la Membrana/metabolismo , Miofibroblastos/metabolismo , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Receptores de Factores de Crecimiento Transformadores beta , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral , Antígeno B7-H1
6.
Nature ; 593(7860): 575-579, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33981032

RESUMEN

Fibroblasts are non-haematopoietic structural cells that define the architecture of organs, support the homeostasis of tissue-resident cells and have key roles in fibrosis, cancer, autoimmunity and wound healing1. Recent studies have described fibroblast heterogeneity within individual tissues1. However, the field lacks a characterization of fibroblasts at single-cell resolution across tissues in healthy and diseased organs. Here we constructed fibroblast atlases by integrating single-cell transcriptomic data from about 230,000 fibroblasts across 17 tissues, 50 datasets, 11 disease states and 2 species. Mouse fibroblast atlases and a DptIRESCreERT2 knock-in mouse identified two universal fibroblast transcriptional subtypes across tissues. Our analysis suggests that these cells can serve as a reservoir that can yield specialized fibroblasts across a broad range of steady-state tissues and activated fibroblasts in disease. Comparison to an atlas of human fibroblasts from perturbed states showed that fibroblast transcriptional states are conserved between mice and humans, including universal fibroblasts and activated phenotypes associated with pathogenicity in human cancer, fibrosis, arthritis and inflammation. In summary, a cross-species and pan-tissue approach to transcriptomics at single-cell resolution has identified key organizing principles of the fibroblast lineage in health and disease.


Asunto(s)
Fibroblastos/citología , Transcriptoma , Animales , Células Cultivadas , Enfermedad , Femenino , Fibroblastos/clasificación , Técnicas de Sustitución del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias , Especificidad de Órganos , Fenotipo , RNA-Seq , Análisis de la Célula Individual , Células del Estroma
7.
Nature ; 591(7848): 131-136, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33472215

RESUMEN

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.


Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Muerte Celular , Membrana Celular/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Animales , Apoptosis , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/genética , Muerte Celular/genética , Femenino , Humanos , Macrófagos , Masculino , Ratones , Mutación , Necrosis , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/genética , Multimerización de Proteína , Piroptosis/genética
8.
Nature ; 587(7833): 275-280, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32971525

RESUMEN

Mutations in the death receptor FAS1,2 or its ligand FASL3 cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome4-6. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses7-12, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages13 was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.


Asunto(s)
Caspasa 8/metabolismo , Citocinas/inmunología , Inmunidad Innata/inmunología , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Células Cultivadas , Citocinas/antagonistas & inhibidores , Humanos , Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo
9.
Nature ; 574(7778): 428-431, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31511692

RESUMEN

The aspartate-specific cysteine protease caspase-8 suppresses necroptotic cell death mediated by RIPK3 and MLKL. Indeed, mice that lack caspase-8 die in a RIPK3- and MLKL-dependent manner during embryogenesis1-3. In humans, caspase-8 deficiency is associated with immunodeficiency4 or very early onset inflammatory bowel disease5. The substrates that are cleaved by caspase-8 to prevent necroptosis in vivo have not been defined. Here we show that knock-in mice that express catalytically inactive caspase-8(C362A) die as embryos owing to MLKL-dependent necroptosis, similar to caspase-8-deficient mice. Thus, caspase-8 must cleave itself, other proteins or both to inhibit necroptosis. Mice that express caspase-8(D212A/D218A/D225A/D387A), which cannot cleave itself, were viable, as were mice that express c-FLIP or CYLD proteins that had been mutated to prevent cleavage by caspase-8. By contrast, mice that express RIPK1(D325A), in which the caspase-8 cleavage site Asp325 had been mutated, died mid-gestation. Embryonic lethality was prevented by inactivation of RIPK1, loss of TNFR1, or loss of both MLKL and the caspase-8 adaptor FADD, but not by loss of MLKL alone. Thus, RIPK1(D325A) appears to trigger cell death mediated by TNF, the kinase activity of RIPK1 and FADD-caspase-8. Accordingly, dying endothelial cells that contain cleaved caspase-3 were abnormally abundant in yolk sacs of Ripk1D325A/D325A embryos. Heterozygous Ripk1D325A/+ cells and mice were viable, but were also more susceptible to TNF-induced cell death than were wild-type cells or mice. Our data show that Asp325 of RIPK1 is essential for limiting aberrant cell death in response to TNF, consistent with the idea that cleavage of RIPK1 by caspase-8 is a mechanism for dismantling death-inducing complexes.


Asunto(s)
Apoptosis/fisiología , Caspasa 8/metabolismo , Necroptosis/fisiología , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Desarrollo Embrionario/genética , Humanos , Ratones , Mutación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética
10.
Nature ; 575(7784): 679-682, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31723262

RESUMEN

Caspase-8 is a protease with both pro-death and pro-survival functions: it mediates apoptosis induced by death receptors such as TNFR11, and suppresses necroptosis mediated by the kinase RIPK3 and the pseudokinase MLKL2-4. Mice that lack caspase-8 display MLKL-dependent embryonic lethality4, as do mice that express catalytically inactive CASP8(C362A)5. Casp8C362A/C362AMlkl-/- mice die during the perinatal period5, whereas Casp8-/-Mlkl-/- mice are viable4, which indicates that inactive caspase-8 also has a pro-death scaffolding function. Here we show that mutant CASP8(C362A) induces the formation of ASC (also known as PYCARD) specks, and caspase-1-dependent cleavage of GSDMD and caspases 3 and 7 in MLKL-deficient mouse intestines around embryonic day 18. Caspase-1 and its adaptor ASC contributed to the perinatal lethal phenotype because a number of Casp8C362A/C362AMlkl-/-Casp1-/- and Casp8C362A/C362AMlkl-/-Asc-/- mice survived beyond weaning. Transfection studies suggest that inactive caspase-8 adopts a distinct conformation to active caspase-8, enabling its prodomain to engage ASC. Upregulation of the lipopolysaccharide sensor caspase-11 in the intestines of both Casp8C362A/C362AMlkl-/- and Casp8C362A/C362AMlkl-/-Casp1-/- mice also contributed to lethality because Casp8C362A/C362AMlkl-/-Casp1-/-Casp11-/- (Casp11 is also known as Casp4) neonates survived more often than Casp8C362A/C362AMlkl-/-Casp1-/- neonates. Finally, Casp8C362A/C362ARipk3-/-Casp1-/-Casp11-/- mice survived longer than Casp8C362A/C362AMlkl-/-Casp1-/-Casp11-/- mice, indicating that a necroptosis-independent function of RIPK3 also contributes to lethality. Thus, unanticipated plasticity in death pathways is revealed when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.


Asunto(s)
Caspasa 8/metabolismo , Muerte Celular/genética , Mucosa Intestinal/citología , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 8/genética , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Mucosa Intestinal/enzimología , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo
11.
Nature ; 574(7777): 249-253, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31578523

RESUMEN

The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells1. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality2-5. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6-/- skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.


Asunto(s)
Diferenciación Celular , Células Epidérmicas/citología , Epidermis/fisiología , Factores Reguladores del Interferón/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Anomalías Múltiples/genética , Animales , Labio Leporino/genética , Fisura del Paladar/genética , Quistes/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Células Epidérmicas/metabolismo , Epidermis/embriología , Anomalías del Ojo/genética , Femenino , Dedos/anomalías , Regulación de la Expresión Génica , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/genética , Rodilla/anomalías , Articulación de la Rodilla/anomalías , Labio/anomalías , Metabolismo de los Lípidos/genética , Deformidades Congénitas de las Extremidades Inferiores/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Sindactilia/genética , Anomalías Urogenitales/genética
12.
J Immunol ; 208(12): 2632-2642, 2022 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-35675956

RESUMEN

Genetic and environmental cues shape the evolution of the B cell Ig repertoire. Activation-induced cytidine deaminase (AID) is essential to generating Ig diversity through isotype class switching and somatic mutations, which then directly influence clonal selection. Impaired B cell development in AID-knockout mice has made it difficult to study Ig diversification in an aging repertoire. Therefore, in this report, we used a novel inducible AID-knockout mouse model and discovered that deleting AID in adult mice caused spontaneous germinal center formation. Deep sequencing of the IgH repertoire revealed that Ab diversification begins early in life and evolves over time. Our data suggest that activated B cells form germinal centers at steady state and facilitate continuous diversification of the B cell repertoire. In support, we identified shared B cell lineages that were class switched and showed age-dependent rates of mutation. Our data provide novel context to the genesis of the B cell repertoire that may benefit the understanding of autoimmunity and the strength of an immune response to infection.


Asunto(s)
Citidina Desaminasa , Cambio de Clase de Inmunoglobulina , Animales , Linfocitos B , Citidina Desaminasa/genética , Centro Germinal , Cambio de Clase de Inmunoglobulina/genética , Ratones , Ratones Noqueados , Hipermutación Somática de Inmunoglobulina
13.
Nature ; 559(7712): 120-124, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29950720

RESUMEN

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease1,2 and embryonic lethality during mouse development3. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.


Asunto(s)
Muerte Celular , Enzimas Desubicuitinizantes/metabolismo , Endopeptidasas/metabolismo , Inflamación/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinación , Animales , Caspasa 8/genética , Caspasa 8/metabolismo , Muerte Celular/genética , Enzimas Desubicuitinizantes/genética , Pérdida del Embrión/genética , Endopeptidasas/genética , Inflamación/enzimología , Inflamación/genética , Interferón Tipo I/biosíntesis , Ratones , Ratones Endogámicos C57BL , 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 , Ubiquitinación/genética , Pérdida de Peso/genética
14.
Toxicol Appl Pharmacol ; 475: 116627, 2023 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-37453479

RESUMEN

SMARCA2 and SMARCA4 are the ATPases of the SWI/SNF chromatin remodeling complex, which play a significant role in regulating transcriptional activity and DNA repair in cells. SMARCA2 has become an appealing synthetic-lethal, therapeutic target in oncology, as mutational loss of SMARCA4 in many cancers leads to a functional dependency on residual SMARCA2 activity. Thus, for therapeutic development, an important step is understanding any potential safety target-associated liabilities of SMARCA2 inhibition. To best mimic a SMARCA2 therapeutic, a tamoxifen-inducible (TAMi) conditional knockout (cKO) rat was developed using CRISPR technology to understand the safety profile of Smarca2 genetic ablation in a model system that avoids potential juvenile and developmental phenotypes. As the rat is the prototypical rodent species utilized in toxicology studies, a comprehensive toxicological and pathological assessment was conducted in both heterozygote and homozygous knockout rats at timepoints up to 28 days, alongside relevant corresponding controls. To our knowledge, this represents the first TAMi cKO rat model utilized for safety assessment evaluations. No significant target-associated phenotypes were observed when Smarca2 was ablated in mature (11- to 15-week-old) rats; however subsequent induction of SMARCA4 was evident that could indicate potential compensatory activity. Similar to mouse models, rat CreERT2-transgene and TAMi toxicities were characterized to avoid confounding study interpretation. In summary, a lack of significant safety findings in Smarca2 cKO rats highlights the potential for therapeutics targeting selective SMARCA2 ATPase activity; such therapies are predicted to be tolerated in patients without eliciting significant on-target toxicities.


Asunto(s)
Neoplasias , Tamoxifeno , Ratones , Ratas , Animales , Tamoxifeno/toxicidad , Adenosina Trifosfatasas , Mutación
15.
J Allergy Clin Immunol ; 150(4): 972-978.e7, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35487308

RESUMEN

BACKGROUND: Clinical studies of type 2 (T2) cytokine-related neutralizing antibodies in asthma have identified a substantial subset of patients with low levels of T2 inflammation who do not benefit from T2 cytokine neutralizing antibody treatment. Non-T2 mechanisms are poorly understood in asthma but represent a redefined unmet medical need. OBJECTIVE: We sought to gain a better understanding of genetic contributions to T2-low asthma. METHODS: We utilized an unbiased genome-wide association study of patients with moderate to severe asthma stratified by T2 serum biomarker periostin. We also performed additional expression and biological analysis for the top genetic hits. RESULTS: We identified a novel protective single nucleotide polymorphism at chr19q13.41, which is selectively associated with T2-low asthma and establishes Kallikrein-related peptidase 5 (KLK5) as the causal gene mediating this association. Heterozygous carriers of the single nucleotide polymorphisms have reduced KLK5 expression. KLK5 is secreted by human bronchial epithelial cells and elevated in asthma bronchial alveolar lavage. T2 cytokines IL-4 and IL-13 downregulate KLK5 in human bronchial epithelial cells. KLK5, dependent on its catalytic function, induces epithelial chemokine/cytokine expression. Finally, overexpression of KLK5 in airway or lack of an endogenous KLK5 inhibitor, SPINK5, leads to spontaneous airway neutrophilic inflammation. CONCLUSION: Our data identify KLK5 to be the causal gene at a novel locus at chr19q13.41 associated with T2-low asthma.


Asunto(s)
Asma , Estudio de Asociación del Genoma Completo , Anticuerpos Neutralizantes/genética , Asma/genética , Quimiocinas/genética , Citocinas/metabolismo , Humanos , Inflamación/genética , Interleucina-13/genética , Interleucina-4/genética , Calicreínas/genética , Calicreínas/metabolismo
16.
Mol Biol Rep ; 49(4): 3281-3288, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-35107736

RESUMEN

INTRODUCTION: Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. For example, enhanced green fluorescent protein or Cre recombinase is placed under the control of endogenous genes to define promoter expression patterns. METHODS AND RESULTS: The most important step in the process is to demonstrate that a gene targeting vector is correctly integrated in the genome at the desired chromosomal location. The rapid identification of correctly targeted ES cell clones is facilitated by proper targeting vector construction, rapid screening procedures, and advances in cell culture. Here, we optimized and functionally linked magnetic activated cell sorting (MACS) technology as well as multiplex droplet digital PCR (ddPCR) to our ES cell screening process to achieve a greater than 60% assurance that ES clones are correctly targeted. In a further refinement of the process, drug selection cassettes are removed from ES cells with adenovirus technology. We describe this improved workflow and illustrate the reduction in time between therapeutic target identification and experimental validation. CONCLUSION: In sum, we describe a novel and effective implementation of ddPCR, multiMACS, and adenovirus recombinase into a streamlined screening workflow that significantly reduces timelines for gene targeting in mouse ES cells.


Asunto(s)
Células Madre Embrionarias , Vectores Genéticos , Alelos , Animales , Células Madre Embrionarias/metabolismo , Marcación de Gen/métodos , Vectores Genéticos/genética , Genotipo , Ratones
17.
Nature ; 540(7631): 129-133, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27819682

RESUMEN

Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1RHIM/RHIM mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but previously had no defined role in development, also prevented perinatal lethality in Ripk1RHIM/RHIM mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1RHIM/RHIMMlkl-/- macrophages, but not in wild-type, Mlkl-/- or Ripk1RHIM/RHIMRipk3RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.


Asunto(s)
Apoptosis , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/metabolismo , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Secuencias de Aminoácidos , Animales , Animales Recién Nacidos , Caspasa 8/genética , Caspasa 8/metabolismo , Embrión de Mamíferos/citología , Femenino , Glicoproteínas/química , Glicoproteínas/deficiencia , Macrófagos/metabolismo , Masculino , Ratones , Mutación , Fosforilación , Unión Proteica , Proteínas Quinasas/deficiencia , Proteínas Quinasas/metabolismo , Proteínas de Unión al ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Factor de Necrosis Tumoral alfa/farmacología
18.
Am J Physiol Cell Physiol ; 320(2): C162-C174, 2021 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-33206546

RESUMEN

Proteolytic processing of procollagens is a central step during collagen fibril formation. Bone morphogenic protein 1 (BMP1) is a metalloprotease that plays an important role in the cleavage of carboxy-terminal (COOH-terminal) propeptides from procollagens. Although the removal of propeptides is required to generate mature collagen fibrils, the contribution of BMP1 to this proteolytic process and its action site remain to be fully determined. In this study, using postnatal lung fibroblasts as a model system, we showed that genetic ablation of Bmp1 in primary murine lung fibroblasts abrogated COOH-terminal cleavage from type I procollagen as measured by COOH-terminal propeptide of type I procollagen (CICP) production. We also showed that inhibition of BMP1 by siRNA-mediated knockdown or small-molecule inhibitor reduced the vast majority of CICP production and collagen deposition in primary human lung fibroblasts. Furthermore, we discovered and characterized two antibody inhibitors for BMP1. In both postnatal lung fibroblast and organoid cultures, BMP1 blockade prevented CICP production. Together, these findings reveal a nonredundant role of extracellular BMP1 to process CICP in lung fibroblasts and suggest that development of antibody inhibitors is a viable pharmacological approach to target BMP1 proteinase activity in fibrotic diseases.


Asunto(s)
Proteína Morfogenética Ósea 1/metabolismo , Líquido Extracelular/metabolismo , Fibroblastos/metabolismo , Pulmón/metabolismo , Fragmentos de Péptidos/metabolismo , Procolágeno/metabolismo , Proteolisis , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 1/antagonistas & inhibidores , Proteína Morfogenética Ósea 1/genética , Células CHO , Cricetinae , Cricetulus , Líquido Extracelular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Células HEK293 , Humanos , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Organoides , Oxadiazoles/farmacología , Fragmentos de Péptidos/genética , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Procolágeno/genética , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , Conejos
19.
Nat Methods ; 15(7): 512-514, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29786090

RESUMEN

Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ingeniería Genética , Genómica/métodos , Animales , Línea Celular , Femenino , Masculino , Ratones , Reacción en Cadena de la Polimerasa Multiplex/métodos , ARN/genética , Ratas , Secuenciación Completa del Genoma/métodos
20.
Nature ; 526(7575): 666-71, 2015 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-26375259

RESUMEN

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1ß processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1ß maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1ß secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasas/metabolismo , Inflamasomas/metabolismo , Transducción de Señal , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Caspasas Iniciadoras , Línea Celular , Femenino , Bacterias Gramnegativas/inmunología , Humanos , Inflamasomas/efectos de los fármacos , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Mutación/genética , Necrosis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a Fosfato , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Sepsis/microbiología , Transducción de Señal/genética , Análisis de Supervivencia
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