RESUMEN
Tissue immunity and responses to injury depend on the coordinated action and communication among physiological systems. Here, we show that, upon injury, adaptive responses to the microbiota directly promote sensory neuron regeneration. At homeostasis, tissue-resident commensal-specific T cells colocalize with sensory nerve fibers within the dermis, express a transcriptional program associated with neuronal interaction and repair, and promote axon growth and local nerve regeneration following injury. Mechanistically, our data reveal that the cytokine interleukin-17A (IL-17A) released by commensal-specific Th17 cells upon injury directly signals to sensory neurons via IL-17 receptor A, the transcription of which is specifically upregulated in injured neurons. Collectively, our work reveals that in the context of tissue damage, preemptive immunity to the microbiota can rapidly bridge biological systems by directly promoting neuronal repair, while also identifying IL-17A as a major determinant of this fundamental process.
Asunto(s)
Interleucina-17 , Microbiota , Regeneración Nerviosa , Células Th17 , Axones , Regeneración Nerviosa/fisiología , Células Receptoras Sensoriales , Animales , Ratones , Células Th17/citologíaRESUMEN
Regulatory T cells (Treg cells) can activate multiple suppressive mechanisms in vitro after activation via the T cell antigen receptor, resulting in antigen-independent suppression. However, it remains unclear whether similar pathways operate in vivo. Here we found that antigen-specific Treg cells activated by dendritic cells (DCs) pulsed with two antigens suppressed conventional naive T cells (Tnaive cells) specific for both cognate antigens and non-cognate antigens in vitro but suppressed only Tnaive cells specific for cognate antigen in vivo. Antigen-specific Treg cells formed strong interactions with DCs, resulting in selective inhibition of the binding of Tnaive cells to cognate antigen yet allowing bystander Tnaive cell access. Strong binding resulted in the removal of the complex of cognate peptide and major histocompatibility complex class II (pMHCII) from the DC surface, reducing the capacity of DCs to present antigen. The enhanced binding of Treg cells to DCs, coupled with their capacity to deplete pMHCII, represents a novel pathway for Treg cell-mediated suppression and may be a mechanism by which Treg cells maintain immune homeostasis.
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Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Tolerancia Inmunológica/inmunología , Linfocitos T Reguladores/inmunología , Animales , Efecto Espectador/inmunología , Células Cultivadas , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos/inmunología , Cultivo Primario de Células , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
The oropharyngeal mucosa serves as a perpetual pathogen entry point and a critical site for viral replication and spread. Here, we demonstrate that type 1 innate lymphoid cells (ILC1s) were the major immune force providing early protection during acute oral mucosal viral infection. Using intravital microscopy, we show that ILC1s populated and patrolled the uninfected labial mucosa. ILC1s produced interferon-γ (IFN-γ) in the absence of infection, leading to the upregulation of key antiviral genes, which were downregulated in uninfected animals upon genetic ablation of ILC1s or antibody-based neutralization of IFN-γ. Thus, tonic IFN-γ production generates increased oral mucosal viral resistance even before infection. Our results demonstrate barrier-tissue protection through tissue surveillance in the absence of rearranged-antigen receptors and the induction of an antiviral state during homeostasis. This aspect of ILC1 biology raises the possibility that these cells do not share true functional redundancy with other tissue-resident lymphocytes.
Asunto(s)
Interferón gamma/metabolismo , Linfocitos/inmunología , Orofaringe/inmunología , Mucosa Respiratoria/inmunología , Virus Vaccinia/fisiología , Vaccinia/inmunología , Animales , Células Cultivadas , Resistencia a la Enfermedad , Humanos , Inmunidad Innata , Interferón gamma/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Dominio T Box/genética , Células TH1/inmunologíaRESUMEN
The transcriptional programs that guide lymphocyte differentiation depend on the precise expression and timing of transcription factors (TFs). The TF BACH2 is essential for T and B lymphocytes and is associated with an archetypal super-enhancer (SE). Single-nucleotide variants in the BACH2 locus are associated with several autoimmune diseases, but BACH2 mutations that cause Mendelian monogenic primary immunodeficiency have not previously been identified. Here we describe a syndrome of BACH2-related immunodeficiency and autoimmunity (BRIDA) that results from BACH2 haploinsufficiency. Affected subjects had lymphocyte-maturation defects that caused immunoglobulin deficiency and intestinal inflammation. The mutations disrupted protein stability by interfering with homodimerization or by causing aggregation. We observed analogous lymphocyte defects in Bach2-heterozygous mice. More generally, we observed that genes that cause monogenic haploinsufficient diseases were substantially enriched for TFs and SE architecture. These findings reveal a previously unrecognized feature of SE architecture in Mendelian diseases of immunity: heterozygous mutations in SE-regulated genes identified by whole-exome/genome sequencing may have greater significance than previously recognized.
Asunto(s)
Enfermedades Autoinmunes/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Síndromes de Inmunodeficiencia/genética , Corticoesteroides/uso terapéutico , Adulto , Enfermedades Autoinmunes/complicaciones , Colitis/complicaciones , Colitis/genética , Colitis/patología , Femenino , Fiebre/complicaciones , Fiebre/tratamiento farmacológico , Fiebre/genética , Haploinsuficiencia , Heterocigoto , Humanos , Síndromes de Inmunodeficiencia/complicaciones , Linfopenia/complicaciones , Linfopenia/genética , Masculino , Persona de Mediana Edad , Mutación , Pancitopenia/complicaciones , Pancitopenia/tratamiento farmacológico , Pancitopenia/genética , Linaje , Polimorfismo de Nucleótido Simple , Recurrencia , Infecciones del Sistema Respiratorio/complicaciones , Infecciones del Sistema Respiratorio/diagnóstico por imagen , Infecciones del Sistema Respiratorio/genética , Esplenomegalia/complicaciones , Esplenomegalia/genética , Síndrome , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
The somatosensory nervous system surveils external stimuli at barrier tissues, regulating innate immune cells under infection and inflammation. The roles of sensory neurons in controlling the adaptive immune system, and more specifically immunity to the microbiota, however, remain elusive. Here, we identified a mechanism for direct neuroimmune communication between commensal-specific T lymphocytes and somatosensory neurons mediated by the neuropeptide calcitonin gene-related peptide (CGRP) in the skin. Intravital imaging revealed that commensal-specific T cells are in close proximity to cutaneous nerve fibers in vivo. Correspondingly, we observed upregulation of the receptor for the neuropeptide CGRP, RAMP1, in CD8+ T lymphocytes induced by skin commensal colonization. The neuroimmune CGRP-RAMP1 signaling axis functions in commensal-specific T cells to constrain Type 17 responses and moderate the activation status of microbiota-reactive lymphocytes at homeostasis. As such, modulation of neuroimmune CGRP-RAMP1 signaling in commensal-specific T cells shapes the overall activation status of the skin epithelium, thereby impacting the outcome of responses to insults such as wounding. The ability of somatosensory neurons to control adaptive immunity to the microbiota via the CGRP-RAMP1 axis underscores the various layers of regulation and multisystem coordination required for optimal microbiota-reactive T cell functions under steady state and pathology.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina , Neuroinmunomodulación , Péptido Relacionado con Gen de Calcitonina/genética , Proteína 1 Modificadora de la Actividad de Receptores/genética , Receptores de Péptido Relacionado con el Gen de Calcitonina , Inmunidad AdaptativaRESUMEN
The establishment of type 2 responses driven by allergic sensitization prior to exposure to helminth parasites has demonstrated how tissue-specific responses can protect against migrating larval stages, but, as a consequence, allow for immune-mediated, parasite/allergy-associated morbidity. In this way, whether helminth cross-reacting allergen-specific antibodies are produced and play a role during the helminth infection, or exacerbate the allergic outcome awaits elucidation. Thus, the main objective of the study was to investigate whether house dust mite (HDM) sensitization triggers allergen-specific antibodies that interact with Ascaris antigens and mediate antibody-dependent deleterious effects on these parasites as well as, to assess the capacity of cross-reactive helminth proteins to trigger allergic inflammation in house dust mite presensitized mice. Here, we show that the sensitization with HDM-extract drives marked IgE and IgG1 antibody responses that cross-react with Ascaris larval antigens. Proteomic analysis of Ascaris larval antigens recognized by these HDM-specific antibodies identified Ascaris tropomyosin and enolase as the 2 major HDM homologues based on high sequence and structural similarity. Moreover, the helminth tropomyosin could drive Type-2 associated pulmonary inflammation similar to HDM following HDM tropomyosin sensitization. The HDM-triggered IgE cross-reactive antibodies were found to be functional as they mediated immediate hypersensitivity responses in skin testing. Finally, we demonstrated that HDM sensitization in either B cells or FcγRIII alpha-chain deficient mice indicated that the allergen driven cell-mediated larval killing is not antibody-dependent. Taken together, our data suggest that aeroallergen sensitization drives helminth reactive antibodies through molecular and structural similarity between HDM and Ascaris antigens suggesting that cross-reactive immune responses help drive allergic inflammation.
Asunto(s)
Polvo/inmunología , Hipersensibilidad/inmunología , Pyroglyphidae/inmunología , Animales , Antígenos Dermatofagoides/inmunología , Proteínas del Helminto/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Ratones , ProteómicaRESUMEN
BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) represents the fastest growing underlying cause of hepatocellular carcinoma (HCC) and has been shown to impact immune effector cell function. The standard of care for the treatment of advanced HCC is immune checkpoint inhibitor (ICI) therapy, yet NASH may negatively affect the efficacy of ICI therapy in HCC. The immunologic mechanisms underlying the impact of NASH on ICI therapy remain unclear. METHODS: Herein, using multiple murine NASH models, we analysed the influence of NASH on the CD8+ T-cell-dependent anti-PD-1 responses against liver cancer. We characterised CD8+ T cells' transcriptomic, functional, and motility changes in mice receiving a normal diet (ND) or a NASH diet. RESULTS: NASH blunted the effect of anti-PD-1 therapy against liver cancers in multiple murine models. NASH caused a proinflammatory phenotypic change of hepatic CD8+ T cells. Transcriptomic analysis revealed changes related to NASH-dependent impairment of hepatic CD8+ T-cell metabolism. In vivo imaging analysis showed reduced motility of intratumoural CD8+ T cells. Metformin treatment rescued the efficacy of anti-PD-1 therapy against liver tumours in NASH. CONCLUSIONS: We discovered that CD8+ T-cell metabolism is critically altered in the context of NASH-related liver cancer, impacting the effectiveness of ICI therapy - a finding which has therapeutic implications in patients with NASH-related liver cancer. LAY SUMMARY: Non-alcoholic steatohepatitis represents the fastest growing cause of hepatocellular carcinoma. It is also associated with reduced efficacy of immunotherapy, which is the standard of care for advanced hepatocellular carcinoma. Herein, we show that non-alcoholic steatohepatitis is associated with impaired motility, metabolic function, and response to anti-PD-1 treatment in hepatic CD8+ T cells, which can be rescued by metformin treatment.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Metformina , Enfermedad del Hígado Graso no Alcohólico , Animales , Linfocitos T CD8-positivos/metabolismo , Carcinoma Hepatocelular/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Hígado/patología , Neoplasias Hepáticas/etiología , Metformina/farmacología , Metformina/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from those initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the "Trojan Horse" model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite.
Asunto(s)
Insectos Vectores/parasitología , Leishmania major/fisiología , Leishmaniasis Cutánea/parasitología , Phlebotomus/parasitología , Animales , Dermis/inmunología , Dermis/parasitología , Femenino , Citometría de Flujo , Leishmaniasis Cutánea/patología , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Neutrófilos/inmunología , Neutrófilos/parasitología , FagocitosisRESUMEN
House dust mite (HDM) extract is a common trigger of asthma in humans. Chronic exposure to HDM also induces asthma-like pathology in mice. Allergic responses to HDM and other allergens are linked to release of IL-25, IL-33, and TSLP by epithelial cells; these cytokines, especially IL-33, target innate lymphoid cells type 2 to produce type 2 cytokines. To what extent and by what mechanisms IL-25 contributes to chronic HDM-induced pathology is not well understood. In humans, elevated levels of IL-25 appear to be associated with cases of uncontrolled asthma and exacerbated attacks. In this article, we demonstrate that blockade of IL-25 signaling in either lung conventional dendritic cells or in T cells resulted in similar decreases in production of IL-13 and IL-9 by T cells, reduced mast cell accumulation and tissue remodeling, and improved lung function but had only modest effects on eosinophilia. Stimulation of conventional dendritic cells by IL-25 promoted proximal accumulation of Th cells, and stimulation of Th cells by IL-25 locally promoted IL-13 and IL-9 production. IL-25 made notable contributions to chronic HDM-induced allergic asthma pathology by facilitating clustering and cross-stimulation of different cell types in tissue. Therapeutic targeting of IL-25 in combination with other treatments may be beneficial.
Asunto(s)
Asma/inmunología , Células Dendríticas/inmunología , Interleucinas/inmunología , Pyroglyphidae/inmunología , Células Th2/inmunología , Animales , Asma/patología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/fisiología , Adenosina Difosfato/metabolismo , Animales , Línea Celular Tumoral , Activación Enzimática , Humanos , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Traumatismos Experimentales por Radiación/enzimología , Transducción de Señal , Timo/enzimología , Timo/efectos de la radiaciónRESUMEN
B cells express the innate receptor, TLR9, which signals in response to unmethylated CpG sequences in microbial DNA. Of the two major classes of CpG-containing oligonucleotides, CpG-A appears restricted to inducing type 1 IFN in innate immune cells and CpG-B to activating B cells to proliferate and produce Abs and inflammatory cytokines. Although CpGs are candidates for adjuvants to boost innate and adaptive immunity, our understanding of the effect of CpG-A and CpG-B on B cell responses is incomplete. In this study we show that both CpG-B and CpG-A activated B cells in vitro to proliferate, secrete Abs and IL-6, and that neither CpG-B nor CpG-A alone induced type 1 IFN production. However, when incorporated into the cationic lipid, DOTAP, CpG-A, but not CpG-B, induced a type 1 IFN response in B cells in vitro and in vivo. We provide evidence that differences in the function of CpG-A and CpG-B may be related to their intracellular trafficking in B cells. These findings fill an important gap in our understanding of the B cell response to CpGs, with implications for the use of CpG-A and CpG-B as immunomodulators.
Asunto(s)
Linfocitos B/inmunología , Interferón Tipo I/biosíntesis , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/inmunología , Animales , Formación de Anticuerpos , Linfocitos B/efectos de los fármacos , Cationes/inmunología , Citocinas/genética , Citocinas/inmunología , Inmunidad Innata , Factores Inmunológicos/metabolismo , Interferón Tipo I/inmunología , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Lípidos/administración & dosificación , Lípidos/química , Lípidos/farmacología , Activación de Linfocitos , Ratones , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/farmacología , Receptor Toll-Like 9/agonistasRESUMEN
The incidence of pulmonary nontuberculous mycobacteria (NTM) disease is increasing, but host responses in respiratory epithelium infected with NTM are not fully understood. In this work, we aimed to identify infection-relevant gene expression signatures of NTM infection of the respiratory epithelium. We infected air-liquid interface (ALI) primary respiratory epithelial cell cultures with Mycobacterium avium subsp. avium (MAC) or Mycobacterium abscessus subsp. abscessus (MAB). We used cells from four different donors to obtain generalizable data. Differentiated respiratory epithelial cells at the ALI were infected with MAC or MAB at a multiplicity of infection of 100:1 or 1,000:1, and RNA sequencing was performed at Days 1 and 3 after infection. In response to infection, we found down-regulation of ciliary genes but upregulation of genes associated with cytokines/chemokines, such as IL-32, and cholesterol biosynthesis. Inflammatory response genes tended to be more upregulated by MAB than by MAC infection. Primary respiratory epithelial cell infection with NTM at the ALI identified ciliary function, cholesterol biosynthesis, and cytokine/chemokine production as major host responses to infection. Some of these pathways may be amenable to therapeutic manipulation.
Asunto(s)
Colesterol/biosíntesis , Células Epiteliales/metabolismo , Infecciones por Mycobacterium no Tuberculosas/inmunología , Micobacterias no Tuberculosas/inmunología , Mucosa Respiratoria/metabolismo , Adulto , Anciano , Adhesión Bacteriana/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Células Epiteliales/microbiología , Femenino , Perfilación de la Expresión Génica , Humanos , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Interleucinas/biosíntesis , Interleucinas/inmunología , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Masculino , Persona de Mediana Edad , Infecciones por Mycobacterium no Tuberculosas/genética , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mucosa Respiratoria/citología , Mucosa Respiratoria/microbiologíaRESUMEN
Skin infection with the poxvirus vaccinia (VV) elicits a powerful, inflammatory cellular response that clears virus infection in a coordinated, spatially organized manner. Given the high concentration of pro-inflammatory effectors at areas of viral infection, it is unclear how tissue pathology is limited while virus-infected cells are being eliminated. To better understand the spatial dynamics of the anti-inflammatory response to a cutaneous viral infection, we first screened cytokine mRNA expression levels after epicutaneous (ec.) VV infection and found a large increase the anti-inflammatory cytokine IL-10. Ex vivo analyses revealed that T cells in the skin were the primary IL-10-producing cells. To understand the distribution of IL-10-producing T cells in vivo, we performed multiphoton intravital microscopy (MPM) of VV-infected mice, assessing the location and dynamic behavior of IL-10 producing cells. Although virus-specific T cells were distributed throughout areas of the inflamed skin lacking overt virus-infection, IL-10+ cells closely associated with large keratinocytic foci of virus replication where they exhibited similar motility patterns to bulk antigen-specific CD8+ T cells. Paradoxically, neutralizing secreted IL-10 in vivo with an anti-IL-10 antibody increased viral lesion size and viral replication. Additional analyses demonstrated that IL-10 antibody administration decreased recruitment of CCR2+ inflammatory monocytes, which were important for reducing viral burden in the infected skin. Based upon these findings, we conclude that spatially concentrated IL-10 production limits cutaneous viral replication and dissemination, likely through modulation of the innate immune repertoire at the site of viral growth.
Asunto(s)
Interleucina-10/metabolismo , Virus Vaccinia/inmunología , Vaccinia/inmunología , Animales , Citocinas/genética , Citocinas/metabolismo , Femenino , Interleucina-10/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/inmunología , Piel/inmunología , Organismos Libres de Patógenos Específicos , Linfocitos T/inmunología , Vaccinia/virología , Replicación ViralRESUMEN
Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF-ß1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.
Asunto(s)
Inmunidad Humoral/inmunología , Ganglios Linfáticos/inmunología , Infiltración Neutrófila/inmunología , Infecciones Estafilocócicas/inmunología , Traslado Adoptivo , Animales , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Staphylococcus aureus/inmunologíaRESUMEN
Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a highly evolutionarily conserved cytosolic protein initially identified in Caenorhabditis elegans, where it was assigned a regulatory role in asymmetric cell divisions. It functions as a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13 and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes in embryonic stem cell lines. To test its role in hematopoiesis and B lymphocytes specifically, we generated ric8 (fl/fl) vav1-cre and ric8 (fl/fl) mb1-cre mice. The major hematopoietic cell lineages developed in the ric8 (fl/fl) vav1-cre mice, notwithstanding severe reduction in Gαi2/3, Gαq, and Gα13 proteins. B lymphocyte-specific loss of Ric-8A did not compromise bone marrow B lymphopoiesis, but splenic marginal zone B cell development failed, and B cells underpopulated lymphoid organs. The ric8 (fl/fl) mb1-cre B cells exhibited poor responses to chemokines, abnormal trafficking, improper in situ positioning, and loss of polarity components during B cell differentiation. The ric8 (fl/fl) mb1-cre mice had a severely disrupted lymphoid architecture and poor primary and secondary Ab responses. In B lymphocytes, Ric-8A is essential for normal Gα protein levels and is required for B cell differentiation, trafficking, and Ab responses.
Asunto(s)
Linfocitos B/inmunología , Subunidad alfa de la Proteína de Unión al GTP Gi2/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/inmunología , Factores de Intercambio de Guanina Nucleótido/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Animales , Linfocitos B/metabolismo , Western Blotting , Calcio/inmunología , Calcio/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Centro Germinal/inmunología , Centro Germinal/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Inmunidad Humoral/genética , Inmunidad Humoral/inmunología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/inmunología , Proteínas Proto-Oncogénicas c-vav/metabolismo , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/metabolismo , Imagen de Lapso de TiempoRESUMEN
To determine whether leukocytes need to open endothelial cell contacts during extravasation, we decided to generate mice with strongly stabilized endothelial junctions. To this end, we replaced VE-cadherin genetically by a VE-cadherin-α-catenin fusion construct. Such mice were completely resistant to the induction of vascular leaks by VEGF or histamine. Neutrophil or lymphocyte recruitment into inflamed cremaster, lung and skin were strongly inhibited in these mice, documenting the importance of the junctional route in vivo. Surprisingly, lymphocyte homing into lymph nodes was not inhibited. VE-cadherin-α-catenin associated more intensely with the actin cytoskeleton as demonstrated by its membrane mobility and detergent extractability. Our results establish the junctional route as the main pathway for extravasating leukocytes in several, although not in all tissues. Furthermore, in these tissues, plasticity of the VE-cadherin-catenin complex is central for the leukocyte diapedesis mechanism.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar , Leucocitos/fisiología , Migración Transendotelial y Transepitelial , alfa Catenina/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Antígenos CD/genética , Cadherinas/genética , Células Cultivadas , Endotelio Vascular/metabolismo , Femenino , Técnicas de Sustitución del Gen , Inflamación/genética , Inflamación/metabolismo , Uniones Intercelulares/metabolismo , Leucocitos/metabolismo , Pulmón/metabolismo , Ganglios Linfáticos/metabolismo , Masculino , Ratones , Músculo Esquelético/metabolismo , Piel/metabolismo , alfa Catenina/genéticaRESUMEN
B lymphocyte recirculation through lymph nodes (LNs) requires crossing endothelial barriers and chemoattractant-triggered cell migration. Here we show how LN anatomy and chemoattractant receptor signaling organize B lymphocyte LN trafficking. Blood-borne B cells predominately used CCR7 signaling to adhere to high endothelial venules (HEVs). New B cell emigrants slowly transited the HEV perivenule space, and thereafter localized nearby, avoiding the follicle. Eventually, the newly arrived B cells entered the basal portion of the follicle gradually populating it. In contrast, newly arriving activated B cells rapidly crossed HEVs and migrated toward the lymph node follicle. During their LN residency, recirculating B cells reacquired their sphingosine-1 phospate receptor 1 (S1P1) receptors and markedly attenuated their sensitivity to chemokines. Eventually, the B cells exited the LN follicle by entering the cortical lymphatics or returning to the paracortical cords. Upon entering the lymph, the B cells lost their polarity, down-regulated their S1P1 receptors, and subsequently strongly up-regulated their sensitivity to chemokines. These results are summarized in a model of homeostatic trafficking of B cells through LNs.
Asunto(s)
Linfocitos B/inmunología , Quimiocinas/metabolismo , Regulación hacia Abajo , Ganglios Linfáticos/citología , Sistema Linfático/anatomía & histología , Receptores de Quimiocina/metabolismo , Transducción de Señal , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Adhesión Celular , Células Cultivadas , Quimiotaxis de Leucocito , Cruzamientos Genéticos , Ingle , Cinética , Ganglios Linfáticos/anatomía & histología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Sistema Linfático/citología , Sistema Linfático/inmunología , Sistema Linfático/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Receptores CCR7/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Migración Transendotelial y TransepitelialRESUMEN
Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.
Asunto(s)
Células Presentadoras de Antígenos , Autoinmunidad , Epítopos , Comunicación Celular , CisteínaRESUMEN
Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.
RESUMEN
Tissue-resident macrophages (TRMs) are critical for tissue homeostasis/repair. We previously showed that dermal TRMs produce CCL24 (eotaxin2) which mediates their interaction with IL-4 producing eosinophils, required to maintain their number and M2-like properties in the TH1 environment of the Leishmania major infected skin. Here, we unveil another layer of TRM self-maintenance involving their production of TSLP, an alarmin typically characterized as epithelial cell-derived. Both TSLP signaling and IL-5+ innate lymphoid cell 2 (ILC2s) were shown to maintain the number of dermal TRMs and promote infection. Single cell RNA sequencing identified the dermal TRMs as the sole source of TSLP and CCL24. Development of Ccl24-cre mice permitted specific labeling of dermal TRMs, as well as interstitial TRMs from other organs. Genetic ablation of TSLP from dermal TRMs reduced the number of dermal TRMs, and disease was ameliorated. Thus, by orchestrating localized type 2 circuitries with ILC2s and eosinophils, dermal TRMs are self-maintained as a replicative niche for L. major.