RESUMO
Adipose tissue plasticity is orchestrated by molecularly and functionally diverse cells within the stromal vascular fraction (SVF). Although several mouse and human adipose SVF cellular subpopulations have by now been identified, we still lack an understanding of the cellular and functional variability of adipose stem and progenitor cell (ASPC) populations across human fat depots. To address this, we performed single-cell and bulk RNA sequencing (RNA-seq) analyses of >30 SVF/Lin- samples across four human adipose depots, revealing two ubiquitous human ASPC (hASPC) subpopulations with distinct proliferative and adipogenic properties but also depot- and BMI-dependent proportions. Furthermore, we identified an omental-specific, high IGFBP2-expressing stromal population that transitions between mesothelial and mesenchymal cell states and inhibits hASPC adipogenesis through IGFBP2 secretion. Our analyses highlight the molecular and cellular uniqueness of different adipose niches, while our discovery of an anti-adipogenic IGFBP2+ omental-specific population provides a new rationale for the biomedically relevant, limited adipogenic capacity of omental hASPCs.
Assuntos
Adipogenia , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina , Omento , Células Estromais , Humanos , Omento/metabolismo , Omento/citologia , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Células Estromais/metabolismo , Células Estromais/citologia , Feminino , Masculino , Pessoa de Meia-Idade , Tecido Adiposo/metabolismo , Tecido Adiposo/citologia , Adulto , Epitélio/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Idoso , AnimaisRESUMO
Gut-draining mesenteric and celiac lymph nodes (mLNs and celLNs) critically contribute to peripheral tolerance toward food and microbial antigens by supporting the de novo induction of regulatory T cells (Tregs). These tolerogenic properties of mLNs and celLNs are stably imprinted within stromal cells (SCs) by microbial signals and vitamin A (VA), respectively. Here, we report that a single, transient gastrointestinal infection in the neonatal, but not adult, period durably abrogates the efficient Treg-inducing capacity of celLNs by altering the subset composition and gene expression profile of celLNSCs. These cells carry information about the early-life pathogen encounter until adulthood and durably instruct migratory dendritic cells entering the celLN with reduced tolerogenic properties. Mechanistically, transiently reduced VA levels cause long-lasting celLN functional impairment, which can be rescued by early-life treatment with VA. Together, our data highlight the therapeutic potential of VA to prevent sequelae post gastrointestinal infections in infants.
Assuntos
Linfonodos , Linfócitos T Reguladores , Vitamina A , Animais , Linfonodos/imunologia , Linfonodos/patologia , Linfonodos/efeitos dos fármacos , Vitamina A/farmacologia , Vitamina A/uso terapêutico , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Camundongos , Animais Recém-Nascidos , Tolerância Imunológica/efeitos dos fármacos , Células Dendríticas/imunologia , Camundongos Endogâmicos C57BL , FemininoRESUMO
The opportunistic pathogen Pseudomonas aeruginosa causes antibiotic-recalcitrant pneumonia by forming biofilms in the respiratory tract. Despite extensive in vitro experimentation, how P. aeruginosa forms biofilms at the airway mucosa is unresolved. To investigate the process of biofilm formation in realistic conditions, we developed AirGels: 3D, optically accessible tissue-engineered human lung models that emulate the airway mucosal environment. AirGels recapitulate important factors that mediate host-pathogen interactions including mucus secretion, flow and air-liquid interface (ALI), while accommodating high-resolution live microscopy. With AirGels, we investigated the contributions of mucus to P. aeruginosa biofilm biogenesis in in vivo-like conditions. We found that P. aeruginosa forms mucus-associated biofilms within hours by contracting luminal mucus early during colonization. Mucus contractions facilitate aggregation, thereby nucleating biofilms. We show that P. aeruginosa actively contracts mucus using retractile filaments called type IV pili. Our results therefore suggest that, while protecting epithelia, mucus constitutes a breeding ground for biofilms.
Assuntos
Biofilmes , Pseudomonas aeruginosa , Humanos , Antibacterianos/farmacologia , Muco , PulmãoRESUMO
Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9+ FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19+ and Cxcl9+ FRCs in LNs of Irf3-/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs.
Assuntos
Linfonodos , Células Estromais , Camundongos , Animais , Camundongos Endogâmicos C57BL , Células Estromais/metabolismo , Linfonodos/patologia , Moléculas de Adesão Celular/metabolismo , Antígenos CD34/metabolismoRESUMO
Acute graft versus host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) inflicted by alloreactive T cells primed in secondary lymphoid organs (SLOs) and subsequent damage to aGvHD target tissues. In recent years, Treg transfer and/or expansion has emerged as a promising therapy to modulate aGvHD. However, cellular niches essential for fostering Tregs to prevent aGvHD have not been explored. Here, we tested whether and to what extent MHC class II (MHCII) expressed on Ccl19+ fibroblastic reticular cells (FRCs) shape the donor CD4+ T cell response during aGvHD. Animals lacking MHCII expression on Ccl19-Cre-expressing FRCs (MHCIIΔCcl19) showed aberrant CD4+ T cell activation in the effector phase, resulting in exacerbated aGvHD that was associated with significantly reduced expansion of Foxp3+ Tregs and invariant NK T (iNKT) cells. Skewed Treg maintenance in MHCIIΔCcl19 mice resulted in loss of protection from aGvHD provided by adoptively transferred donor Tregs. In contrast, although FRCs upregulated costimulatory surface receptors, and although they degraded and processed exogenous antigens after myeloablative irradiation, FRCs were dispensable to activate alloreactive CD4+ T cells in 2 mouse models of aGvHD. In summary, these data reveal an immunoprotective, MHCII-mediated function of FRC niches in secondary lymphoid organs (SLOs) after allo-HCT and highlight a framework of cellular and molecular interactions that regulate CD4+ T cell alloimmunity.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Camundongos , Animais , Linfócitos T Reguladores , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/métodosRESUMO
Conventional dendritic cells (cDCs) arise from committed precursor dendritic cells (pre-DCs) in the bone marrow that continuously seed the periphery. Pre-DCs and other upstream progenitors proliferate and mature in response to Fms-related receptor tyrosine kinase 3 ligand, which is considered the key cytokine for cDC development. However, other cytokines such as stem cell factor and colony-stimulating factor 1 (CSF1) were also shown to induce pre-DC maturation into DC-like cells. Yet, it is still only incompletely understood which cells contribute to cDC development once pre-DCs arrive in peripheral tissues. Here, we analysed the impact of lymph node (LN) fibroblastic stromal cells (FSCs) on the maturation of pre-DCs into cDC-like cells. We could demonstrate that ex vivo isolated LN FSCs co-cultured with pre-DCs induce precursor maturation into DC-like cells, which were capable of efficiently promoting the proliferation of naïve CD4+ T cells. Interestingly, FSCs isolated from distinct LNs induced DC-like cells with highly comparable transcriptomes, characterized by the expression of signature genes of both ex vivo isolated DCs and macrophages. Finally, by performing supplementation and receptor blocking studies, we could demonstrate that CSF1 is a driving factor for LN FSC-mediated pre-DC maturation into DC-like cells. In summary, we could identify CSF1 as a stromal cell-derived factor that has the potential to support the maturation of pre-DCs into cDC-like cells within secondary lymphoid organs.
Assuntos
Células Dendríticas , Fator Estimulador de Colônias de Macrófagos , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/metabolismo , Linfonodos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Células Estromais , Linfócitos TRESUMO
Non-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100 kb. These molecularly coordinated regions are named "variable chromatin modules" (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect a VCM-modulating noncoding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a large change in TF binding activity and chromatin state at an enhancer cluster spanning >150 kb, coinciding with subtle, long-range chromatin compaction and robust AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology.
Assuntos
Cromatina , Leucemia Linfocítica Crônica de Células B , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Fatores de Transcrição/metabolismoRESUMO
Single-cell RNA sequencing (scRNA-seq) approaches have transformed our ability to resolve cellular properties across systems, but are currently tailored toward large cell inputs (>1,000 cells). This renders them inefficient and costly when processing small, individual tissue samples, a problem that tends to be resolved by loading bulk samples, yielding confounded mosaic cell population read-outs. Here, we developed a deterministic, mRNA-capture bead and cell co-encapsulation dropleting system, DisCo, aimed at processing low-input samples (<500 cells). We demonstrate that DisCo enables precise particle and cell positioning and droplet sorting control through combined machine-vision and multilayer microfluidics, enabling continuous processing of low-input single-cell suspensions at high capture efficiency (>70%) and at speeds up to 350 cells per hour. To underscore DisCo's unique capabilities, we analyzed 31 individual intestinal organoids at varying developmental stages. This revealed extensive organoid heterogeneity, identifying distinct subtypes including a regenerative fetal-like Ly6a+ stem cell population that persists as symmetrical cysts, or spheroids, even under differentiation conditions, and an uncharacterized 'gobloid' subtype consisting predominantly of precursor and mature (Muc2+) goblet cells. To complement this dataset and to demonstrate DisCo's capacity to process low-input, in vivo-derived tissues, we also analyzed individual mouse intestinal crypts. This revealed the existence of crypts with a compositional similarity to spheroids, which consisted predominantly of regenerative stem cells, suggesting the existence of regenerating crypts in the homeostatic intestine. These findings demonstrate the unique power of DisCo in providing high-resolution snapshots of cellular heterogeneity in small, individual tissues.
Assuntos
Organoides , Análise de Célula Única , Animais , Diferenciação Celular , Mucosa Intestinal , Camundongos , Células-TroncoRESUMO
Our understanding of the composition and functions of splenic stromal cells remains incomplete. Here, based on analysis of over 20,000 single cell transcriptomes of splenic fibroblasts, we characterized the phenotypic and functional heterogeneity of these cells in healthy state and during virus infection. We describe eleven transcriptionally distinct fibroblastic cell clusters, reassuring known subsets and revealing yet unascertained heterogeneity amongst fibroblasts occupying diverse splenic niches. We further identify striking differences in innate immune signatures of distinct stromal compartments in vivo. Compared to other fibroblasts and to endothelial cells, Ly6C+ fibroblasts of the red pulp were selectively endowed with enhanced interferon-stimulated gene expression in homeostasis, upon systemic interferon stimulation and during virus infection in vivo. Collectively, we provide an updated map of fibroblastic cell diversity in the spleen that suggests a specialized innate immune function for splenic red pulp fibroblasts.
Assuntos
Fibroblastos/metabolismo , Infecções por Herpesviridae/virologia , Imunidade Inata , Transcriptoma , Animais , Feminino , Fibroblastos/imunologia , Homeostase , Masculino , Camundongos , Muromegalovirus/fisiologia , Análise de Célula Única , Baço/imunologia , Baço/metabolismoRESUMO
Mesenteric lymph nodes (mLNs) are sentinel sites of enteral immunosurveillance and immune homeostasis. Immune cells from the gastrointestinal tract (GIT) are constantly recruited to the mLNs in steady-state and under inflammatory conditions resulting in the induction of tolerance and immune cells activation, respectively. Surgical dissection and transplantation of lymph nodes (LN) is a technique that has supported seminal work to study LN function and is useful to investigate resident stromal and endothelial cell biology and their cellular interactions in experimental disease models. Here, we provide a detailed protocol of syngeneic mLN transplantation and report assays to analyze effective mLN engraftment in congenic recipients. Transplanted mLNs allow to study T cell activation and proliferation in preclinical mouse models. Donor mLNs proved viable and functional after surgical transplantation and regenerated blood and lymphatic vessels. Immune cells from the host completely colonized the transplanted mLNs within 7-8 weeks after the surgical intervention. After allogeneic hematopoietic cell transplantation (allo-HCT), adoptively transferred allogeneic CD4+ T cells from FVB/N (H-2q) mice homed to the transplanted mLNs in C57BL/6 (H-2b) recipients during the initiation phase of acute graft-versus-host disease (aGvHD). These CD4+ T cells retained full proliferative capacity and upregulated effector and gut homing molecules comparable to those in mLNs from unmanipulated wild-type recipients. Wild type mLNs transplanted into MHCII deficient syngeneic hosts sufficed to activate alloreactive T cells upon allogeneic hematopoietic cell transplantation, even in the absence of MHCII+ CD11c+ myeloid cells. These data support that orthotopically transplanted mLNs maintain physiological functions after transplantation. The technique of LN transplantation can be applied to study migratory and resident cell compartment interactions in mLNs as well as immune reactions from and to the gut under inflammatory and non-inflammatory conditions.
Assuntos
Diferenciação Celular , Proliferação de Células , Doença Enxerto-Hospedeiro/imunologia , Linfonodos/transplante , Ativação Linfocitária , Linfócitos T/transplante , Doença Aguda , Transferência Adotiva , Animais , Modelos Animais de Doenças , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/metabolismo , Transplante de Células-Tronco Hematopoéticas , Antígenos de Histocompatibilidade Classe II/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Mesentério , Camundongos Endogâmicos C57BL , Fenótipo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tolerância ao Transplante , Transplante IsogênicoRESUMO
Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus-host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Viral da Expressão Gênica/genética , SARS-CoV-2/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Animais , Antivirais/farmacologia , Células Cultivadas , Chlorocebus aethiops , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Interferons/farmacologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Especificidade da Espécie , Temperatura , Células Vero , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Intestinal Foxp3+ regulatory T cell (Treg) subsets are crucial players in tolerance to microbiota-derived and food-borne antigens, and compelling evidence suggests that the intestinal microbiota modulates their generation, functional specialization, and maintenance. Selected bacterial species and microbiota-derived metabolites, such as short-chain fatty acids (SCFAs), have been reported to promote Treg homeostasis in the intestinal lamina propria. Furthermore, gut-draining mesenteric lymph nodes (mLNs) are particularly efficient sites for the generation of peripherally induced Tregs (pTregs). Despite this knowledge, the direct role of the microbiota and their metabolites in the early stages of pTreg induction within mLNs is not fully elucidated. Here, using an adoptive transfer-based pTreg induction system, we demonstrate that neither transfer of a dysbiotic microbiota nor dietary SCFA supplementation modulated the pTreg induction capacity of mLNs. Even mice housed under germ-free (GF) conditions displayed equivalent pTreg induction within mLNs. Further molecular characterization of these de novo induced pTregs from mLNs by dissection of their transcriptomes and accessible chromatin regions revealed that the microbiota indeed has a limited impact and does not contribute to the initialization of the Treg-specific epigenetic landscape. Overall, our data suggest that the microbiota is dispensable for the early stages of pTreg induction within mLNs.
Assuntos
Microbioma Gastrointestinal/imunologia , Linfonodos/imunologia , Mesentério/imunologia , Linfócitos T Reguladores/imunologia , Animais , Cromatina/metabolismo , Disbiose/microbiologia , Disbiose/patologia , Epigênese Genética/efeitos dos fármacos , Ácidos Graxos Voláteis/farmacologia , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Perfilação da Expressão Gênica , Linfonodos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Hydrodynamic-based microfluidic platforms enable single-cell arraying and analysis over time. Despite the advantages of established microfluidic systems, long-term analysis and proliferation of cells selected in such devices require off-chip recovery of cells as well as an investigation of on-chip analysis on cell phenotype, requirements still largely unmet. Here, we introduce a device for single-cell isolation, selective retrieval and off-chip recovery. To this end, singularly addressable three-dimensional electrodes are embedded within a microfluidic channel, allowing the selective release of single cells from their trapping site through application of a negative dielectrophoretic (DEP) force. Selective capture and release are carried out in standard culture medium and cells can be subsequently mitigated towards a recovery well using micro-engineered hybrid SU-8/PDMS pneumatic valves. Importantly, transcriptional analysis of recovered cells revealed only marginal alteration of their molecular profile upon DEP application, underscored by minor transcriptional changes induced upon injection into the microfluidic device. Therefore, the established microfluidic system combining targeted DEP manipulation with downstream hydrodynamic coordination of single cells provides a powerful means to handle and manipulate individual cells within one device.
RESUMO
Foxp3+ regulatory T cells (Tregs) co-expressing the Th17-lineage specification factor RORγt represent a unique Treg subpopulation that has been reported to be induced upon response to gut microbiota within the intestinal immune system. Hence, RORγt+ Tregs are considered to solely consist of peripherally induced Foxp3+ Tregs (pTregs), and the possibility that also thymus-derived Treg (tTregs) can upregulate RORγt expression and contribute to the pool of RORγt+ Tregs was largely ignored. Here, we expand our knowledge on the origin of RORγt+ Tregs by demonstrating that also tTregs can attain RORγt expression. In transgenic Foxp3∆CNS1-Cre reporter mice, a substantial fraction of CNS1-independent Tregs, predominantly consisting of tTregs, was found to co-express RORγt. In addition, genuine tTregs isolated from thymi of Foxp3hCD2RAGGFP reporter mice initiated RORγt expression both in vitro and in vivo, particularly under inflammatory conditions. In conclusion, our data demonstrate that tTregs can upregulate RORγt expression under inflammatory conditions and that hence RORγt+ Tregs can be regarded as a heterogeneous population consisting of both pTregs and tTregs. KEY MESSAGES: RORγt cannot be considered as a marker for pTregs. CNS1-independent tTregs within the colon display RORγt expression. RORγt can be induced in genuine tTregs, particularly under inflammatory conditions. RORγt+ Tregs are a heterogeneous population consisting of both pTregs and tTregs.
Assuntos
Fatores de Transcrição Forkhead/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Colite/imunologia , Colite/terapia , Inflamação/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T Reguladores/transplante , Regulação para CimaRESUMO
Gut-draining mesenteric lymph nodes (mLNs) are important for inducing peripheral tolerance towards food and commensal antigens by providing an optimal microenvironment for de novo generation of Foxp3+ regulatory T cells (Tregs). We previously identified microbiota-imprinted mLN stromal cells as a critical component in tolerance induction. Here we show that this imprinting process already takes place in the neonatal phase, and renders the mLN stromal cell compartment resistant to inflammatory perturbations later in life. LN transplantation and single-cell RNA-seq uncover stably imprinted expression signatures in mLN fibroblastic stromal cells. Subsetting common stromal cells across gut-draining mLNs and skin-draining LNs further refine their location-specific immunomodulatory functions, such as subset-specific expression of Aldh1a2/3. Finally, we demonstrate that mLN stromal cells shape resident dendritic cells to attain high Treg-inducing capacity in a Bmp2-dependent manner. Thus, crosstalk between mLN stromal and resident dendritic cells provides a robust regulatory mechanism for the maintenance of intestinal tolerance.
Assuntos
Células Dendríticas/imunologia , Tolerância Imunológica/imunologia , Linfonodos/imunologia , Células Estromais/imunologia , Animais , Animais Recém-Nascidos , Microambiente Celular/genética , Microambiente Celular/imunologia , Células Dendríticas/metabolismo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Tolerância Imunológica/genética , Linfonodos/metabolismo , Linfonodos/transplante , Mesentério/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Estromais/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
Yersinia pseudotuberculosis is a Gram-negative bacterium and zoonotic pathogen responsible for a wide range of diseases, ranging from mild diarrhea, enterocolitis, lymphatic adenitis to persistent local inflammation. The Y. pseudotuberculosis invasin D (InvD) molecule belongs to the invasin (InvA)-type autotransporter proteins, but its structure and function remain unknown. In this study, we present the first crystal structure of InvD, analyzed its expression and function in a murine infection model, and identified its target molecule in the host. We found that InvD is induced at 37 °C and expressed in vivo 2-4 days after infection, indicating that InvD is a virulence factor. During infection, InvD was expressed in all parts of the intestinal tract, but not in deeper lymphoid tissues. The crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold that, apart from the canonical ß-sheets, comprises various modifications of and insertions into the Ig-core structure. We identified the Fab fragment of host-derived IgG/IgA antibodies as the target of the adhesion domain. Phage display panning and flow cytometry data further revealed that InvD exhibits a preferential binding specificity toward antibodies with VH3/VK1 variable domains and that it is specifically recruited to a subset of B cells. This finding suggests that InvD modulates Ig functions in the intestine and affects direct interactions with a subset of cell surface-exposed B-cell receptors. In summary, our results provide extensive insights into the structure of InvD and its specific interaction with the target molecule in the host.
Assuntos
Adesinas Bacterianas/metabolismo , Anticorpos/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Intestinos/microbiologia , Infecções por Yersinia pseudotuberculosis/microbiologia , Yersinia pseudotuberculosis/patogenicidade , Adesinas Bacterianas/química , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Aderência Bacteriana , Feminino , Fragmentos Fab das Imunoglobulinas/imunologia , Intestinos/imunologia , Intestinos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Virulência , Yersinia pseudotuberculosis/imunologia , Infecções por Yersinia pseudotuberculosis/metabolismo , Infecções por Yersinia pseudotuberculosis/patologiaRESUMO
Adaptive immunity is essentially required to control acute infection with enteropathogenic Yersinia pseudotuberculosis (Yptb). We have recently demonstrated that Yptb can directly modulate naïve CD4+ T cell differentiation. However, whether fully differentiated forkhead box protein P3 (Foxp3+) regulatory T cells (Tregs), fundamental key players to maintain immune homeostasis, are targeted by Yptb remains elusive. Here, we demonstrate that within the CD4+ T cell compartment Yptb preferentially targets Tregs and injects Yersinia outer proteins (Yops) in a process that depends on the type III secretion system and invasins. Remarkably, Yop-translocation into ex vivo isolated Foxp3+ Tregs resulted in a substantial downregulation of Foxp3 expression and a decreased capacity to express the immunosuppressive cytokine interleukin-10 (IL-10). Together, these findings highlight that invasins are critically required to mediate Yptb attachment to Foxp3+ Tregs, which allows efficient Yop-translocation and finally enables the modulation of the Foxp3+ Tregs' suppressive phenotype.
RESUMO
Intestinal regulatory T cells (Tregs) are fundamental in peripheral tolerance toward commensals and food-borne antigens. Accordingly, gut-draining mesenteric lymph nodes (mLNs) represent a site of efficient peripheral de novo Treg induction when compared to skin-draining peripheral LNs (pLNs), and we had recently shown that LN stromal cells substantially contribute to this process. Here, we aimed to unravel the underlying molecular mechanisms and generated immortalized fibroblastic reticular cell lines (iFRCs) from mLNs and pLNs, allowing unlimited investigation of this rare stromal cell subset. In line with our previous findings, mLN-iFRCs showed a higher Treg-inducing capacity when compared to pLN-iFRCs. RNA-seq analysis focusing on secreted molecules revealed a more tolerogenic phenotype of mLN- as compared to pLN-iFRCs. Remarkably, mLN-iFRCs produced substantial numbers of microvesicles (MVs) that carried elevated levels of TGF-ß when compared to pLN-iFRC-derived MVs, and these novel players of intercellular communication were shown to be responsible for the tolerogenic properties of mLN-iFRCs. Thus, stromal cells originating from mLNs contribute to peripheral tolerance by fostering de novo Treg induction using TGF-ß-carrying MVs. This finding provides novel insights into the subcellular/molecular mechanisms of de novo Treg induction and might serve as promising tool for future therapeutic applications to treat inflammatory disorders.
Assuntos
Vesículas Extracelulares/imunologia , Linfonodos/imunologia , Células Estromais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular , Vesículas Extracelulares/genética , Vesículas Extracelulares/ultraestrutura , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica/métodos , Mesentério/imunologia , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Células Estromais/metabolismo , Células Estromais/ultraestrutura , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologiaRESUMO
Adaptive immunity critically contributes to control acute infection with enteropathogenic Yersinia pseudotuberculosis; however, the role of CD4+ T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4+ T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3+ regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within naïve CD4+ T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of naïve CD4+ T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3+ Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4+ T cell subsets by altering their TCR downstream signaling.
Assuntos
Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/microbiologia , Células Th17/microbiologia , Infecções por Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Diferenciação Celular , Feminino , Fatores de Transcrição Forkhead/imunologia , Interações Hospedeiro-Patógeno , Intestinos/imunologia , Intestinos/microbiologia , Camundongos Endogâmicos BALB C , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Yersinia pseudotuberculosis/fisiologiaRESUMO
Background: To successfully limit pathogen dissemination, an immunological link between the entry tissue of the pathogen and the underlying secondary lymphoid organs (SLOs) needs to be established to prime adaptive immune responses. Here, the prerequisite of CCR7 to mount host immune responses within SLOs during gastrointestinal Yersinia pseudotuberculosis infection to limit pathogen spread was investigated. Methods: Survival, bacterial dissemination, and intestinal and systemic pathology of wild-type and CCR7-/- mice were assessed and correlated to the presence of immune cell subsets and cytokine responses throughout the course of infection. Results: The CCR7-/- mice show a significantly higher morbidity and are more prone to pathogen dissemination and intestinal and systemic inflammation during the oral route of infection. Significant impact of CCR7 deficiency over the course of infection on several immunological parameters were observed (ie, elevated neutrophil-dominated innate immune response in Peyer's patches, limited dendritic cell migration to mesenteric lymph nodes [mLNs] causing reduced T cell-mediated adaptive immune responses (in particular Th17-like responses) in mLNs). Conclusions: Our work indicates that CCR7 is required to mount a robust immune response against enteropathogenic Y. pseudotuberculosis by promoting Th17-like responses in mLNs.