RESUMEN
Adaptive immune cell function is regulated by a highly diverse receptor recombined from variable germline-encoded segments that can recognize an almost unlimited array of epitopes. While this diversity enables the recognition of any pathogen, it also poses a risk of self-recognition, leading to autoimmunity. Many layers of regulation are present during both the generation and activation of B cells to prevent this phenomenon, although they are evidently imperfect. In recent years, our ability to analyze immune repertoires at scale has drastically increased, both through advances in sequencing and single-cell analyses. Here, we review the current knowledge on B cell repertoire analyses, focusing on their implication for autoimmunity. These studies demonstrate that a failure of tolerance occurs at multiple independent checkpoints in different autoimmune contexts, particularly during B cell maturation, plasmablast differentiation, and within germinal centers. These failures are marked by distinct repertoire features that may be used to identify disease- or patient-specific therapeutic approaches.
Asunto(s)
Autoinmunidad , Linfocitos B , Tolerancia Inmunológica , Humanos , Autoinmunidad/inmunología , Linfocitos B/inmunología , Animales , Tolerancia Inmunológica/inmunología , Centro Germinal/inmunología , Diferenciación Celular/inmunologíaRESUMEN
Coordinated cell interactions within the esophagus maintain homeostasis, and disruption can lead to eosinophilic esophagitis (EoE), a chronic inflammatory disease with poorly understood pathogenesis. We profile 421,312 individual cells from the esophageal mucosa of 7 healthy and 15 EoE participants, revealing 60 cell subsets and functional alterations in cell states, compositions, and interactions that highlight previously unclear features of EoE. Active disease displays enrichment of ALOX15+ macrophages, PRDM16+ dendritic cells expressing the EoE risk gene ATP10A, and cycling mast cells, with concomitant reduction of TH17 cells. Ligand-receptor expression uncovers eosinophil recruitment programs, increased fibroblast interactions in disease, and IL-9+IL-4+IL-13+ TH2 and endothelial cells as potential mast cell interactors. Resolution of inflammation-associated signatures includes mast and CD4+ TRM cell contraction and cell type-specific downregulation of eosinophil chemoattractant, growth, and survival factors. These cellular alterations in EoE and remission advance our understanding of eosinophilic inflammation and opportunities for therapeutic intervention.
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Esofagitis Eosinofílica , Humanos , Esofagitis Eosinofílica/genética , Esofagitis Eosinofílica/patología , Células Endoteliales/metabolismo , Interleucina-13 , Inflamación/genéticaRESUMEN
Recent studies have characterized various mouse antigen-presenting cells (APCs) expressing the lymphoid-lineage transcription factor RORγt (Retinoid-related orphan receptor gamma t), which exhibit distinct phenotypic features and are implicated in the induction of peripheral regulatory T cells (Tregs) and immune tolerance to microbiota and self-antigens. These APCs encompass Janus cells and Thetis cell subsets, some of which express the AutoImmune REgulator (AIRE). RORγt+ MHCII+ type 3 innate lymphoid cells (ILC3) have also been implicated in the instruction of microbiota-specific Tregs. While RORγt+ APCs have been actively investigated in mice, the identity and function of these cell subsets in humans remain elusive. Herein, we identify a rare subset of RORγt+ cells with dendritic cell (DC) features through integrated single-cell RNA sequencing and single-cell ATAC sequencing. These cells, which we term RORγt+ DC-like cells (R-DC-like), exhibit DC morphology, express the MHC class II machinery, and are distinct from all previously reported DC and ILC3 subsets, but share transcriptional and epigenetic similarities with DC2 and ILC3. We have developed procedures to isolate and expand them in vitro, enabling their functional characterization. R-DC-like cells proliferate in vitro, continue to express RORγt, and differentiate into CD1c+ DC2-like cells. They stimulate the proliferation of allogeneic T cells. The identification of human R-DC-like cells with proliferative potential and plasticity toward CD1c+ DC2-like cells will prompt further investigation into their impact on immune homeostasis, inflammation, and autoimmunity.
Asunto(s)
Inmunidad Innata , Linfocitos , Humanos , Ratones , Animales , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Inflamación/metabolismo , Células DendríticasRESUMEN
In healthy skin, a cutaneous immune system maintains the balance between tolerance towards innocuous environmental antigens and immune responses against pathological agents. In atopic dermatitis (AD), barrier and immune dysfunction result in chronic tissue inflammation. Our understanding of the skin tissue ecosystem in AD remains incomplete with regard to the hallmarks of pathological barrier formation, and cellular state and clonal composition of disease-promoting cells. Here, we generated a multi-modal cell census of 310,691 cells spanning 86 cell subsets from whole skin tissue of 19 adult individuals, including non-lesional and lesional skin from 11 AD patients, and integrated it with 396,321 cells from four studies into a comprehensive human skin cell atlas in health and disease. Reconstruction of human keratinocyte differentiation from basal to cornified layers revealed a disrupted cornification trajectory in AD. This disrupted epithelial differentiation was associated with signals from a unique immune and stromal multicellular community comprised of MMP12 + dendritic cells (DCs), mature migratory DCs, cycling ILCs, NK cells, inflammatory CCL19 + IL4I1 + fibroblasts, and clonally expanded IL13 + IL22 + IL26 + T cells with overlapping type 2 and type 17 characteristics. Cell subsets within this immune and stromal multicellular community were connected by multiple inter-cellular positive feedback loops predicted to impact community assembly and maintenance. AD GWAS gene expression was enriched both in disrupted cornified keratinocytes and in cell subsets from the lesional immune and stromal multicellular community including IL13 + IL22 + IL26 + T cells and ILCs, suggesting that epithelial or immune dysfunction in the context of the observed cellular communication network can initiate and then converge towards AD. Our work highlights specific, disease-associated cell subsets and interactions as potential targets in progression and resolution of chronic inflammation.
RESUMEN
Autophagy is an essential cellular process that is deeply integrated with innate immune signaling; however, studies that examine the impact of autophagic modulation in the context of inflammatory conditions are lacking. Here, using mice with a constitutively active variant of the autophagy gene Beclin1, we show that increased autophagy dampens cytokine production during a model of macrophage activation syndrome and in adherent-invasive Escherichia coli (AIEC) infection. Moreover, loss of functional autophagy through conditional deletion of Beclin1 in myeloid cells significantly enhances innate immunity in these contexts. We further analyzed primary macrophages from these animals with a combination of transcriptomics and proteomics to identify mechanistic targets downstream of autophagy. Our study reveals glutamine/glutathione metabolism and the RNF128/TBK1 axis as independent regulators of inflammation. Altogether, our work highlights increased autophagic flux as a potential approach to reduce inflammation and defines independent mechanistic cascades involved in this control.
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Enfermedad de Crohn , Infecciones por Escherichia coli , Animales , Ratones , Enfermedad de Crohn/metabolismo , Beclina-1/genética , Beclina-1/metabolismo , Autofagia/genética , Macrófagos/metabolismo , Inflamación/metabolismo , Citocinas/metabolismoRESUMEN
The ability of Mycobacterium tuberculosis (Mtb) to establish latency affects disease and response to treatment. The host factors that influence the establishment of latency remain elusive. We engineered a multi-fluorescent Mtb strain that reports survival, active replication, and stressed non-replication states and determined the host transcriptome of the infected macrophages in these states. Additionally, we conducted a genome-wide CRISPR screen to identify host factors that modulated the phenotypic state of Mtb. We validated hits in a phenotype-specific manner and prioritized membrane magnesium transporter 1 (MMGT1) for a detailed mechanistic investigation. Mtb infection of MMGT1-deficient macrophages promoted a switch to persistence, upregulated lipid metabolism genes, and accumulated lipid droplets during infection. Targeting triacylglycerol synthesis reduced both droplet formation and Mtb persistence. The orphan G protein-coupled receptor GPR156 is a key inducer of droplet accumulation in ΔMMGT1 cells. Our work uncovers the role of MMGT1-GPR156-lipid droplets in the induction of Mtb persistence.
Asunto(s)
Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Gotas Lipídicas/metabolismo , Macrófagos/microbiología , Metabolismo de los LípidosRESUMEN
Asthma is a chronic disease most commonly associated with allergy and type 2 inflammation. However, the mechanisms that link airway inflammation to the structural changes that define asthma are incompletely understood. Using a human model of allergen-induced asthma exacerbation, we compared the lower airway mucosa in allergic asthmatics and allergic non-asthmatic controls using single-cell RNA sequencing. In response to allergen, the asthmatic airway epithelium was highly dynamic and up-regulated genes involved in matrix degradation, mucus metaplasia, and glycolysis while failing to induce injury-repair and antioxidant pathways observed in controls. IL9-expressing pathogenic TH2 cells were specific to asthmatic airways and were only observed after allergen challenge. Additionally, conventional type 2 dendritic cells (DC2 that express CD1C) and CCR2-expressing monocyte-derived cells (MCs) were uniquely enriched in asthmatics after allergen, with up-regulation of genes that sustain type 2 inflammation and promote pathologic airway remodeling. In contrast, allergic controls were enriched for macrophage-like MCs that up-regulated tissue repair programs after allergen challenge, suggesting that these populations may protect against asthmatic airway remodeling. Cellular interaction analyses revealed a TH2-mononuclear phagocyte-basal cell interactome unique to asthmatics. These pathogenic cellular circuits were characterized by type 2 programming of immune and structural cells and additional pathways that may sustain and amplify type 2 signals, including TNF family signaling, altered cellular metabolism, failure to engage antioxidant responses, and loss of growth factor signaling. Our findings therefore suggest that pathogenic effector circuits and the absence of proresolution programs drive structural airway disease in response to type 2 inflammation.
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Asma , Hipersensibilidad , Humanos , Antioxidantes , Asma/genética , Alérgenos , InflamaciónRESUMEN
Plasma cells (PCs) constitute a significant fraction of colonic mucosal cells and contribute to inflammatory infiltrates in ulcerative colitis (UC). While gut PCs secrete bacteria-targeting IgA antibodies, their role in UC pathogenesis is unknown. We performed single-cell V(D)J- and RNA-seq on sorted B cells from the colon of healthy individuals and patients with UC. A large fraction of B cell clones is shared between different colon regions, but inflammation in UC broadly disrupts this landscape, causing transcriptomic changes characterized by an increase in the unfolded protein response (UPR) and antigen presentation genes, clonal expansion, and isotype skewing from IgA1 and IgA2 to IgG1. We also directly expressed and assessed the specificity of 152 mAbs from expanded PC clones. These mAbs show low polyreactivity and autoreactivity and instead target both shared bacterial antigens and specific bacterial strains. Altogether, our results characterize the microbiome-specific colon PC response and how its disruption might contribute to inflammation in UC.
Asunto(s)
Colitis Ulcerosa , Humanos , Colitis Ulcerosa/genética , Células Plasmáticas , Colon , Inflamación/metabolismo , Antígenos Bacterianos , Bacterias , Inmunoglobulina A/metabolismo , Mucosa IntestinalRESUMEN
Crohn's disease (CD) is a chronic gastrointestinal disease that is increasing in prevalence worldwide. CD is multifactorial, involving the complex interplay of genetic, immune, and environmental factors, necessitating a system-level understanding of its etiology. To characterize cell-type-specific transcriptional heterogeneity in active CD, we profiled 720,633 cells from the terminal ileum and colon of 71 donors with varying inflammation status. Our integrated datasets revealed organ- and compartment-specific responses to acute and chronic inflammation; most immune changes were in cell composition, whereas transcriptional changes dominated among epithelial and stromal cells. These changes correlated with endoscopic inflammation, but small and large intestines exhibited distinct responses, which were particularly apparent when focusing on IBD risk genes. Finally, we mapped markers of disease-associated myofibroblast activation and identified CHMP1A, TBX3, and RNF168 as regulators of fibrotic complications. Altogether, our results provide a roadmap for understanding cell-type- and organ-specific differences in CD and potential directions for therapeutic development.
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Enfermedad de Crohn , Humanos , Transcriptoma , Colon , Íleon , Inflamación/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
SARS-CoV-2 infection leads to a broad range of outcomes and immune responses, with the development of neutralizing antibodies generally correlated with protection against reinfection. Here, we have characterized both neutralizing activity and T cell responses in a cluster of subjects with mild disease linked to a single spreading event. Surprisingly, we observed sex-specific associations between spike- and particularly nucleoprotein-specific T cell responses and neutralization, with pro-inflammatory cytokines being linked to higher titers only in males. Using single cell immunoprofiling, which provided matched transcriptome and T-cell receptor (TCR) profiles in restimulated CD4 + and CD8 + cells from these subjects, we identified differences in type I IFN signaling that may underlie this difference in antibody generation. Finally, we also identified several TCRs associated with cytokine producing T cells. Altogether, our work maps the breadth of immunological outcomes of SARS-CoV2 infections and highlight the potential role of sex-specific feedback loops during the generation of neutralizing antibodies.
RESUMEN
Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA sequencing (RNA-seq) and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Linfocitos B/metabolismo , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Reacciones Antígeno-Anticuerpo , Linfocitos B/citología , Linfocitos B/virología , COVID-19/patología , COVID-19/virología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Perfilación de la Expresión Génica , Humanos , Inmunoglobulina A/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Dominios Proteicos/inmunología , Multimerización de Proteína , Estructura Cuaternaria de Proteína , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Análisis de Secuencia de ARN , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Chronic itch remains a highly prevalent disorder with limited treatment options. Most chronic itch diseases are thought to be driven by both the nervous and immune systems, but the fundamental molecular and cellular interactions that trigger the development of itch and the acute-to-chronic itch transition remain unknown. Here, we show that skin-infiltrating neutrophils are key initiators of itch in atopic dermatitis, the most prevalent chronic itch disorder. Neutrophil depletion significantly attenuated itch-evoked scratching in a mouse model of atopic dermatitis. Neutrophils were also required for several key hallmarks of chronic itch, including skin hyperinnervation, enhanced expression of itch signaling molecules, and upregulation of inflammatory cytokines, activity-induced genes, and markers of neuropathic itch. Finally, we demonstrate that neutrophils are required for induction of CXCL10, a ligand of the CXCR3 receptor that promotes itch via activation of sensory neurons, and we find that that CXCR3 antagonism attenuates chronic itch.
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Dermatitis Atópica/inmunología , Neutrófilos/inmunología , Prurito/inmunología , Receptores CXCR3/inmunología , Piel/inmunología , Animales , Calcitriol/administración & dosificación , Calcitriol/análogos & derivados , Línea Celular , Quimiocina CXCL10/genética , Quimiocina CXCL10/inmunología , Quimiocina CXCL10/metabolismo , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Dermatitis Atópica/inducido químicamente , Dermatitis Atópica/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Queratinocitos/inmunología , Queratinocitos/metabolismo , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Prurito/inducido químicamente , Prurito/genética , Receptores CXCR3/genética , Receptores CXCR3/metabolismo , Células Receptoras Sensoriales/inmunología , Células Receptoras Sensoriales/metabolismo , Piel/inervación , Piel/metabolismoRESUMEN
The role of the microbiome across a range of physiological process is undebatable, but how this complex community is assembled and regulated remains only partially understood. Recent studies focused on a single sensor show that the neonatal period may represent a critical window and that immune interactions at this time could durably influence the members of the microbiome.
RESUMEN
Signaling by Toll-like receptors (TLRs) on intestinal epithelial cells (IECs) is critical for intestinal homeostasis. To visualize epithelial expression of individual TLRs in vivo, we generated five strains of reporter mice. These mice revealed that TLR expression varied dramatically along the length of the intestine. Indeed, small intestine (SI) IECs expressed low levels of multiple TLRs that were highly expressed by colonic IECs. TLR5 expression was restricted to Paneth cells in the SI epithelium. Intestinal organoid experiments revealed that TLR signaling in Paneth cells or colonic IECs induced a core set of host defense genes, but this set did not include antimicrobial peptides, which instead were induced indirectly by inflammatory cytokines. This comprehensive blueprint of TLR expression and function in IECs reveals unexpected diversity in the responsiveness of IECs to microbial stimuli, and together with the associated reporter strains, provides a resource for further study of innate immunity.
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Colitis/inmunología , Colon/patología , Mucosa Intestinal/fisiología , Intestino Delgado/patología , Células de Paneth/fisiología , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Células Cultivadas , Colitis/inducido químicamente , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Homeostasis , Humanos , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Receptor Cross-Talk , Transducción de Señal , Receptor Toll-Like 5/metabolismoRESUMEN
Although apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. Here we identified macrophage populations that continually engulf ACs in distinct tissues and found that these macrophages share characteristics compatible with immunologically silent clearance of ACs; such characteristics include high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. Removal of the macrophages from tissues resulted in loss of many of these characteristics and the ability to generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment program macrophages for silent AC clearance. The transcription factors KLF2 and KLF4 control the expression of many genes within this AC clearance program. The coordinated expression of AC receptors with genes that limit responses to nucleic acids might ensure maintenance of homeostasis and thus represent a central feature of tissue macrophages.
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Apoptosis , Macrófagos/inmunología , Animales , Femenino , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/fisiología , Activación de Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 9/fisiologíaAsunto(s)
Inmunidad Innata , Moléculas de Patrón Molecular Asociado a Patógenos , Receptores de Reconocimiento de Patrones , Transducción de Señal , Animales , Evolución Biológica , Humanos , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Receptores de Reconocimiento de Patrones/metabolismoRESUMEN
Neutrophils are generally the first immune cells recruited during the development of sterile or microbial inflammation. As these cells express many innate immune receptors with the potential to directly recognize microbial or endogenous signals, we set out to assess whether their functions are locally influenced by the signals present at the onset of inflammation. Using a mouse model of peritonitis, we demonstrate that neutrophils elicited in the presence of C-type lectin receptor ligands have an increased ability to produce cytokines, chemokines, and lipid mediators in response to subsequent TLR stimulation. Importantly, we found that licensing of cytokine production was mediated by paracrine TNF-α-TNFR1 signaling rather than direct ligand sensing, suggesting a form of quorum sensing among neutrophils. Mechanistically, licensing was largely imparted by changes in the posttranscriptional regulation of inflammatory cytokines, whereas production of IL-10 was regulated at the transcriptional level. Altogether, our data suggest that neutrophils rapidly adapt their functions to the local inflammatory milieu. These phenotypic changes may promote rapid neutrophil recruitment in the presence of pathogens but limit inflammation in their absence.