RESUMO
BACKGROUND: Food allergy diagnosis and management causes a number of social and emotional challenges for individuals with food allergies and their caregivers. This has led to increased interest in developing approaches to accurately predict food allergy diagnosis, severity of food allergic reactions, and treatment outcomes. However, the utility of these approaches is somewhat conflicting. OBJECTIVE: We sought to develop and utilize a murine model that mimics the disease course of food allergy diagnosis and treatment in humans and to identify biomarkers that predict reactivity during food challenge (FC) and responsiveness during oral immunotherapy (OIT) and how these outcomes are modified by genetics. METHODS: Skin-sensitized intestinal IL-9 transgenic (IL9Tg) and IL9Tg mice backcrossed onto the IL-4RαY709F background received a single intragastric exposure of egg antigen (ovalbumin), underwent oral FC and OIT; food allergy severity, mast cell activation, and ovalbumin-specific IgE levels were examined to determine the predictability of these outcomes in determining reactivity and treatment outcomes. RESULTS: Subcutaneous sensitization and a single intragastric allergen challenge of egg antigen to BALB/c IL9Tg mice and Il4raY709F IL9Tg induced a food allergic reaction. Enhanced IL-4Rα signaling altered the symptoms induced by the first oral exposure, decreased the cumulative antigen dose, increased the severity of reaction during oral FC, and altered the frequency of adverse events and OIT outcomes. Biomarkers after first oral exposure indicated that only the severity of the initial reaction significantly correlated with cumulative dose of oral FC. CONCLUSION: Collectively, these data indicate that single nucleotide polymorphisms in IL-4Rα can alter clinical symptoms of food allergic reactions, severity, and reactive dose during FC and OIT, and that severity of first reaction can predict the likelihood of reaction during FC in mice with IL-4Rα gain of function.
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Alérgenos , Hipersensibilidade Alimentar , Humanos , Camundongos , Animais , Ovalbumina , Imunoterapia , Camundongos Transgênicos , Biomarcadores , Administração Oral , Dessensibilização ImunológicaRESUMO
INTRODUCTION: Food allergic reactions can be severe and potentially life-threatening and the underlying immunological processes that contribute to the severity of reactions are poorly understood. The aim of this study is to integrate bulk RNA-sequencing of human and mouse peripheral blood mononuclear cells during food allergic reactions and in vivo mouse models of food allergy to identify dysregulated immunological processes associated with severe food allergic reactions. METHODS: Bulk transcriptomics of whole blood from human and mouse following food allergic reactions combined with integrative differential expressed gene bivariate and module eigengene network analyses to identify the whole blood transcriptome associated with food allergy severity. In vivo validation immune cell and gene expression in mice following IgE-mediated reaction. RESULTS: Bulk transcriptomics of whole blood from mice with different severity of food allergy identified gene ontology (GO) biological processes associated with innate and inflammatory immune responses, dysregulation of MAPK and NFkB signalling and identified 429 genes that correlated with reaction severity. Utilizing two independent human cohorts, we identified 335 genes that correlated with severity of peanut-induced food allergic reactions. Mapping mouse food allergy severity transcriptome onto the human transcriptome revealed 11 genes significantly dysregulated and correlated with severity. Analyses of whole blood from mice undergoing an IgE-mediated reaction revealed a rapid change in blood leukocytes particularly inflammatory monocytes (Ly6Chi Ly6G- ) and neutrophils that was associated with changes in CLEC4E, CD218A and GPR27 surface expression. CONCLUSIONS: Collectively, IgE-mediated food allergy severity is associated with a rapid innate inflammatory response associated with acute cellular stress processes and dysregulation of peripheral blood inflammatory myeloid cell frequencies.
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Fenômenos Biológicos , Hipersensibilidade Alimentar , Hipersensibilidade a Amendoim , Humanos , Animais , Camundongos , Leucócitos Mononucleares , Hipersensibilidade Alimentar/genética , Alérgenos , Imunoglobulina E , Receptores Acoplados a Proteínas GRESUMO
BACKGROUND: This study group has previously identified IL-9-producing mucosal mast cell (MMC9) as the primary source of IL-9 to drive intestinal mastocytosis and experimental IgE-mediated food allergy. However, the molecular mechanisms that regulate the expansion of MMC9s remain unknown. OBJECTIVES: This study hypothesized that IL-4 regulates MMC9 development and MMC9-dependent experimental IgE-mediated food allergy. METHODS: An epicutaneous sensitization model was used and bone marrow reconstitution experiments were performed to test the requirement of IL-4 receptor α (IL-4Rα) signaling on MMC9s in experimental IgE-mediated food allergy. Flow cytometric, bulk, and single-cell RNA-sequencing analyses on small intestine (SI) MMC9s were performed to illuminate MMC9 transcriptional signature and the effect of IL-4Rα signaling on MMC9 function. A bone marrow-derived MMC9 culture system was used to define IL-4-BATF signaling in MMC9 development. RESULTS: Epicutaneous sensitization- and bone marrow reconstitution-based models of IgE-mediated food allergy revealed an IL-4 signaling-dependent cell-intrinsic effect on SI MMC9 accumulation and food allergy severity. RNA-sequencing analysis of SI-MMC9s identified 410 gene transcripts reciprocally regulated by IL-4 signaling, including Il9 and Batf. Insilico analyses identified a 3491-gene MMC9 transcriptional signature and identified 2 transcriptionally distinct SI MMC9 populations enriched for metabolic or inflammatory programs. Employing an in vitro MMC9-culture model system showed that generation of MMC9-like cells was induced by IL-4 and this was in part dependent on BATF. CONCLUSIONS: IL-4Rα signaling directly modulates MMC9 function and exacerbation of experimental IgE-mediated food allergic reactions. IL-4Rα regulation of MMC9s is in part BATF-dependent and occurs via modulation of metabolic transcriptional programs.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Hipersensibilidade Alimentar/imunologia , Interleucina-4/imunologia , Interleucina-9/imunologia , Mucosa Intestinal/imunologia , Mastócitos/imunologia , Transdução de Sinais/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Modelos Animais de Doenças , Hipersensibilidade Alimentar/genética , Hipersensibilidade Alimentar/patologia , Interleucina-4/genética , Interleucina-9/genética , Mucosa Intestinal/patologia , Mastócitos/patologia , Camundongos , Camundongos Knockout , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Food allergy (FA) is an increasing problem that has no approved treatment. The pro-TH2 cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) are associated with FA, and mAbs to these cytokines are reported to suppress murine FA development. OBJECTIVE: We sought to determine whether anti-pro-TH2 cytokine mAbs can block both FA maintenance and induction. METHODS: IgE-mediated FA was induced in BALB/c mice by oral gavage with medium-chain triglycerides (MCTs) plus egg white (EW) and was characterized by increased numbers of lamina propria TH2 cells, mast cells, and eosinophils, shock (hypothermia), mast cell degranulation (increased serum mouse mast cell protease 1), increased serum IgG1 anti-EW and IgE levels, and increased IL-4 and IL-13 secretion after MCT/EW challenge. Mice were injected with anti-IL-25, IL-33 receptor, and/or TSLP mAbs before initial oral gavage with MCT/EW to suppress FA development; treatment with the same mAbs was initiated after FA development to suppress established FA. RESULTS: Injection of an mAb to IL-25, IL-33 receptor, or TSLP strongly inhibited FA development. No single mAb to a pro-TH2 cytokine could suppress established FA, and optimal FA suppression required treatment with a cocktail of all 3 anti-pro-TH2 mAbs. Treatment with the 3-mAb cocktail during initial MCT/EW immunization induced EW tolerance. CONCLUSION: All of the pro-TH2 cytokines are required to induce our model of FA, whereas any pro-TH2 cytokine can maintain established FA. Pro-TH2 cytokines prevent oral tolerance. Combined treatment with antagonists to all 3 pro-TH2 cytokines or with an inhibitor of pro-TH2 cytokine production might be able to suppress established human FA.
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Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Neutralizantes/farmacologia , Citocinas/antagonistas & inibidores , Hipersensibilidade Alimentar , Interleucina-33/antagonistas & inibidores , Interleucinas/antagonistas & inibidores , Células Th2/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Neutralizantes/imunologia , Citocinas/imunologia , Feminino , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/patologia , Hipersensibilidade Alimentar/prevenção & controle , Interleucina-33/imunologia , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Células Th2/patologia , Linfopoietina do Estroma do TimoRESUMO
Acute liver failure (ALF) is a potentially life-threatening disorder without any effective treatment strategies. d-Galactosamine (GalN)/lipopolysaccharide (LPS)-induced ALF is a widely used animal model to identify novel hepato-protective agents. In the present study, we investigated the potential of a cannabinoid receptor 2 (CB2) agonist, JWH-133 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran], in the amelioration of GalN/LPS-induced ALF. JWH-133 treatment protected the mice from ALF-associated mortality, mitigated alanine transaminase and proinflammatory cytokines, suppressed histopathological and apoptotic liver damage, and reduced liver infiltration of mononuclear cells (MNCs). Furthermore, JWH-133 pretreatment of M1/M2-polarized macrophages significantly increased the secretion of anti-inflammatory cytokine interleukin-10 (IL-10) in M1 macrophages and potentiated the expression of M2 markers in M2-polarized macrophages. In vivo, JWH-133 treatment also suppressed ALF-triggered expression of M1 markers in liver MNCs, while increasing the expression of M2 markers such as Arg1 and IL-10. microRNA (miR) microarray analysis revealed that JWH-133 treatment altered the expression of only a few miRs in the liver MNCs. Gene ontology analysis of the targets of miRs suggested that Toll-like receptor (TLR) signaling was among the most significantly targeted cellular pathways. Among the altered miRs, miR-145 was found to be the most significantly decreased. This finding correlated with concurrent upregulated expression of its predicted target gene, interleukin-1 receptor-associated kinase 3, a negative regulator of TLR4 signaling. Together, these data are the first to demonstrate that CB2 activation attenuates GalN/LPS-induced ALF by inducing an M1 to M2 shift in macrophages and by regulating the expression of unique miRs that target key molecules involved in the TLR4 pathway.
Assuntos
Galactosamina/efeitos adversos , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , MicroRNAs/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Animais , Apoptose/efeitos dos fármacos , Canabinoides/farmacologia , Citocinas/biossíntese , Regulação para Baixo/efeitos dos fármacos , Feminino , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Falência Hepática Aguda/imunologia , Falência Hepática Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Fenótipo , Receptor CB2 de Canabinoide/agonistas , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
OBJECTIVE: Genomic studies of ovarian cancer (OC) cell lines frequently used in research revealed that these cells do not fully represent high-grade serous ovarian cancer (HGSOC), the most common OC histologic type. However, OC lines that appear to genomically resemble HGSOC have not been extensively used and their growth characteristics in murine xenografts are essentially unknown. METHODS: To better understand growth patterns and characteristics of HGSOC cell lines in vivo, CAOV3, COV362, KURAMOCHI, NIH-OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVSAHO, OVKATE, SNU119 and UWB1.289 cells were assessed for tumor formation in nude mice. Cells were injected intraperitoneally (i.p.) or subcutaneously (s.c.) in female athymic nude mice and allowed to grow (maximum of 90 days) and tumor formation was analyzed. All tumors were sectioned and assessed using H&E staining and immunohistochemistry for p53, PAX8 and WT1 expression. RESULTS: Six lines (OVCAR3, OVCAR4, OVCAR5, OVCAR8, CAOV3, and OVSAHO) formed i.p xenografts with HGSOC histology. OVKATE and COV362 formed s.c. tumors only. Rapid tumor formation was observed for OVCAR3, OVCAR5 and OVCAR8, but only OVCAR8 reliably formed ascites. Tumors derived from OVCAR3, OVCAR4, and OVKATE displayed papillary features. Of the 11 lines examined, three (Kuramochi, SNU119 and UWB1.289) were non-tumorigenic. CONCLUSIONS: Our findings help further define which HGSOC cell models reliably generate tumors and/or ascites, critical information for preclinical drug development, validating in vitro findings, imaging and prevention studies by the OC research community.
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Cistadenocarcinoma Seroso/patologia , Modelos Animais de Doenças , Neoplasias Ovarianas/patologia , Animais , Processos de Crescimento Celular , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/metabolismo , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Gradação de Tumores , Neoplasias Ovarianas/metabolismo , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Proteínas WT1/biossínteseRESUMO
Δ(9)-Tetrahydrocannabinol (THC), the major bioactive component of marijuana, has been shown to induce functional myeloid-derived suppressor cells (MDSCs) in vivo. Here, we studied the involvement of microRNA (miRNA) in this process. CD11b(+)Gr-1(+) MDSCs were purified from peritoneal exudates of mice administered with THC and used for genome-wide miRNA profiling. Expression of CD31 and Ki-67 confirmed that the THC-MDSCs were immature and proliferating. THC-induced MDSCs exhibited distinct miRNA expression signature relative to various myeloid cells and BM precursors. We identified 13 differentially expressed (>2-fold) miRNA in THC-MDSCs relative to control BM precursors. In silico target prediction for these miRNA and pathway analysis using multiple bioinformatics tools revealed significant overrepresentation of Gene Ontology clusters within hematopoiesis, myeloid cell differentiation, and regulation categories. Insulin-like growth factor 1 signaling involved in cell growth and proliferation, and myeloid differentiation pathways were among the most significantly enriched canonical pathways. Among the differentially expressed, miRNA-690 was highly overexpressed in THC-MDSCs (â¼16-fold). Transcription factor CCAAT/enhancer-binding protein α (C/EBPα) was identified as a potential functional target of miR-690. Supporting this, C/EBPα expression was attenuated in THC-MDSCs as compared with BM precursors and exhibited an inverse relation with miR-690. miR-690 knockdown using peptide nucleic acid-antagomiR was able to unblock and significantly increase C/EBPα expression establishing the functional link. Further, CD11b(+)Ly6G(+)Ly6C(+) and CD11b(+)Ly6G(-)Ly6C(+) purified subtypes showed high levels of miR-690 with attenuated C/EBPα expression. Moreover, EL-4 tumor-elicited MDSCs showed increased miR-690 expression. In conclusion, miRNA are significantly altered during the generation of functional MDSC from BM. Select miRNA such as miR-690 targeting genes involved in myeloid expansion and differentiation likely play crucial roles in this process and therefore in cannabinoid-induced immunosuppression.
Assuntos
Analgésicos não Narcóticos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Dronabinol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Tolerância Imunológica/efeitos dos fármacos , Células Mieloides/metabolismo , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/imunologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Tolerância Imunológica/genética , Camundongos , Células Mieloides/citologia , Células Mieloides/imunologiaRESUMO
Mast cells (MCs) are derived from CD34+ hematopoietic progenitors, consist of different subtypes, and are involved in several inflammatory conditions. However, our understanding of human MC developmental trajectories and subtypes has been limited by a scarcity of suitable cellular model systems. Herein, we developed an in vitro model of human MC differentiation from induced pluripotent stem cells (iPSC) to study human MC differentiation trajectories. Flow cytometry characterization of hemopoietic cells derived from the myeloid cells-forming complex (MCFC) revealed an initial increase in Lin- CD34+ hematopoietic progenitors within Weeks 1-3, followed by an increase in CD34- CD45RA- SSClow and SSChigh hematopoietic cells. The Lin- CD34+ hematopoietic progenitors consisted of SSClow CD45RA- CD123± c-Kit+ FcεRI+ populations that were ß7-integrinhigh CD203c+ and ß7-integrinhigh CD203c- cells consistent with CMPFcεRI+ cells. Flow cytometry and cytologic analyses of the CD34- Lin- (SSClow) population revealed hypogranular cell populations, predominantly characterized by CD45RA- CD123± c-Kit+ FcεRI- ß7-integrinlow and CD45RA- CD123± c-Kit- FcεRI+ ß7-integrinMid cells. Analyses of hypergranular SSChigh cells identified Lin- CD34- CD45RA- c-Kit+ FcεRI- and Lin- CD34- CD45RA- c-Kit+ FcεRI+ cells. scRNA-seq analysis of the cells harvested at week 4 of the MCFC culture revealed the presence of monocyte and granulocyte progenitors (n = 547 cells, 26.7 %), Erythrocyte / unknown (n = 85, 4.1 %), neutrophils / myelocytes (n = 211 cells, 10.2 %), mast cell progenitor 1 (n = 599, 29.1 %), mast cell progenitor 2 (n = 152, 7.4 %), committed mast cell precursor (n = 113, 5.5 %), and MCs (n = 353, 17.1 %). In silico analyses of the MC precursor and mature MC populations revealed transcriptionally distinct MC precursor subtype and mature MC states (CMA1+ and CMA1- subtypes). Culturing MC precursor populations in MC maturation media (mast cell media II) led to homogenous mature MC populations as evidenced by high expression of high-affinity IgE receptor, metachromatic granules, presence of MC granule proteins (Tryptase and Chymase) and activation following substance P stimulation and FcεRI crosslinking. This human iPSC-based approach generates MC precursors and phenotypically mature and functional MC populations. This system will be a useful model to generate human MC populations and broaden our understanding of MC biology and transcriptional regulation of MC differentiation trajectories.
RESUMO
Chemical nucleases have found potential applications in the research fields of chemistry, biotechnology and medicine. A variety of metal complexes have been explored as good to outstanding therapeutic agents for DNA cleavage activity most likely via hydrolytic, oxidative or photoinduced cleavage pathways. However, most of these DNA cleaving agents lack their utility in in vivo applications due to their dependence on exogenous oxidants or reductants to achieve successful DNA damage. In view of addressing these issues, the development of metal complexes/organic molecules serving as self-activating chemical nucleases has received growing attention from researchers. In only the last decade, this field has dramatically expanded for the usage of chemical nucleases as therapeutic agents for DNA damage. The present study provides an overview of the opportunities and challenges in the design and development of self-activating chemical nucleases as improved DNA therapeutic candidates in the absence of an external redox agent. The reports on DNA nuclease activity via self-activation, especially with copper, zinc and iron complexes, and their mechanistic investigation have been discussed in this review article.
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Complexos de Coordenação , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Clivagem do DNA , Cobre/química , DNA/química , OxirreduçãoRESUMO
Acute and chronic intestinal inflammation is associated with epithelial damage, resulting in mucosal wounds in the forms of erosions and ulcers in the intestinal tract. Intestinal epithelial cells (IECs) and immune cells in the wound milieu secrete cytokines and lipid mediators to influence repair. Leukotriene B4 (LTB4), a lipid chemokine, binds to its receptor BLT1 and promotes migration of immune cells to sites of active inflammation; however, a role for intestinal epithelial BLT1 during mucosal wound repair is not known. Here we report that BLT1 was expressed in IECs both in vitro and in vivo, where it functioned as a receptor not only for LTB4 but also for another ligand, resolvin E1. Intestinal epithelial BLT1 expression was increased when epithelial cells were exposed to an inflammatory microenvironment. Using human and murine primary colonic epithelial cells, we reveal that the LTB4/BLT1 pathway promoted epithelial migration and proliferation leading to accelerated epithelial wound repair. Furthermore, in vivo intestinal wound repair experiments in BLT1-deficient mice and bone marrow chimeras demonstrated an important contribution of epithelial BLT1 during colonic mucosal wound repair. Taken together, our findings show a potentially novel prorepair in IEC mechanism mediated by BLT1 signaling.
Assuntos
Lipídeos , Humanos , Animais , CamundongosRESUMO
Food allergy is an emerging epidemic, and the underlying mechanisms are not well defined partly due to the lack of robust adjuvant free experimental models of dietary antigen sensitization. As housing mice at thermoneutrality (Tn) - the temperature of metabolic homeostasis (26-30°C) - has been shown to improve modeling various human diseases involved in inflammation, we tested the impact of Tn housing on an experimental model of food sensitization. Here we demonstrate that WT BALB/c mice housed under standard temperature (18-20°C, Ts) conditions translocated the luminal antigens in the small intestine (SI) across the epithelium via goblet cell antigen passages (GAPs). In contrast, food allergy sensitive Il4raF709 mice housed under standard temperature conditions translocated the luminal antigens in the SI across the epithelium via secretory antigen passages (SAPs). Activation of SI antigen passages and oral challenge of Il4raF709 mice with egg allergens at standard temperature predisposed Il4raF709 mice to develop an anaphylactic reaction. Housing Il4raF709 mice at Tn altered systemic type 2 cytokine, IL-4, and the landscape of SI antigen passage patterning (villus and crypt involvement). Activation of SI antigen passages and oral challenge of Il4raF709 mice with egg antigen under Tn conditions led to the robust induction of egg-specific IgE and development of food-induced mast cell activation and hypovolemic shock. Similarly, Tn housing of WT BALB/c mice altered the cellular patterning of SI antigen passage (GAPs to SAPs). Activation of SI antigen passages and the oral challenge of WT BALB/c mice with egg antigen led to systemic reactivity to egg and mast cell activation. Together these data demonstrate that Tn housing alters antigen passage cellular patterning and landscape, and concurrent oral exposure of egg antigens and SAP activation is sufficient to induce oral antigen sensitization.
Assuntos
Alérgenos/metabolismo , Anafilaxia/metabolismo , Hipersensibilidade a Ovo/metabolismo , Proteínas do Ovo/metabolismo , Abrigo para Animais , Intestino Delgado/metabolismo , Temperatura , Administração Oral , Alérgenos/administração & dosagem , Alérgenos/imunologia , Anafilaxia/imunologia , Anafilaxia/microbiologia , Animais , Modelos Animais de Doenças , Hipersensibilidade a Ovo/imunologia , Hipersensibilidade a Ovo/microbiologia , Proteínas do Ovo/administração & dosagem , Proteínas do Ovo/imunologia , Microbioma Gastrointestinal , Células Caliciformes/imunologia , Células Caliciformes/metabolismo , Células Caliciformes/microbiologia , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Knockout , Permeabilidade , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
BACKGROUND & AIMS: CD4+ T cells are regulated by activating and inhibitory cues, and dysregulation of these proper regulatory inputs predisposes these cells to aberrant inflammation and exacerbation of disease. We investigated the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of the CD4+ T-cell inflammatory response and exacerbation of the colitic phenotype. METHODS: We used Il10-/- spontaneous and CD4+CD45RBhi T-cell transfer models of colitis with PIR-B-deficient (Pirb-/-) mice. Flow cytometry, Western blot, and RNA sequencing analysis was performed on wild-type and Pirb-/- CD4+ T cells. In silico analyses were performed on RNA sequencing data set of ileal biopsy samples from pediatric CD and non-inflammatory bowel disease patients and sorted human memory CD4+ T cells. RESULTS: We identified PIR-B expression on memory CD4+ interleukin (IL)17a+ cells. We show that PIR-B regulates CD4+ T-helper 17 cell (Th17)-dependent chronic intestinal inflammatory responses and the development of colitis. Mechanistically, we show that the PIR-B- Src-homology region 2 domain-containing phosphatase-1/2 axis tempers mammalian target of rapamycin complex 1 signaling and mammalian target of rapamycin complex 1-dependent caspase-3/7 apoptosis, resulting in CD4+ IL17a+ cell survival. In silico analyses showed enrichment of transcriptional signatures for Th17 cells (RORC, RORA, and IL17A) and tissue resident memory (HOBIT, IL7R, and BLIMP1) networks in PIR-B+ murine CD4+ T cells and human CD4+ T cells that express the human homologue leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3). High levels of LILRB3 expression were associated strongly with mucosal injury and a proinflammatory Th17 signature, and this signature was restricted to a treatment-naïve, severe pediatric CD population. CONCLUSIONS: Our findings show an intrinsic role for PIR-B/LILRB3 in the regulation of CD4+ IL17a+ T-cell pathogenic memory responses.
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Regulação da Expressão Gênica , Imunomodulação , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Receptores Imunológicos/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Biomarcadores , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Colite/etiologia , Colite/metabolismo , Colite/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Imuno-Histoquímica , Memória Imunológica , Imunofenotipagem , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Receptores Imunológicos/metabolismo , Transdução de SinaisRESUMO
Food allergens are innocuous proteins that promote tolerogenic adaptive immune responses in healthy individuals yet in other individuals induce an allergic adaptive immune response characterized by the presence of antigen-specific immunoglobulin E and type-2 immune cells. The cellular and molecular processes that determine a tolerogenic versus non-tolerogenic immune response to dietary antigens are not fully elucidated. Recently, there have been advances in the identification of roles for microbial communities and anatomical sites of dietary antigen exposure and presentation that have provided new insights into the key regulatory steps in the tolerogenic versus non-tolerogenic decision-making processes. Herein, we will review and discuss recent findings in cellular and molecular processes underlying food sensitization and tolerance, immunological processes underlying severity of food-induced anaphylaxis, and insights obtained from immunotherapy trials.
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Anafilaxia , Hipersensibilidade Alimentar , Microbiota , Alérgenos , Humanos , Tolerância ImunológicaRESUMO
BACKGROUND: Previous studies have revealed an important role for the transcription factor GATA-1 in mast cell maturation and degranulation. However, there have been conflicting reports with respect to the requirement of GATA-1 function in mast cell dependent inflammatory processes. Herein, we examine the requirement of GATA-1 signaling in mast cell effector function and IgE-mast cell-dependent anaphylaxis. OBJECTIVE: To study the requirement of GATA-1 dependent signaling in the development and severity of IgE-mast cell-dependent anaphylaxis in mice. METHODS: Wild type (Balb/c) and mutant ΔdblGata (Balb/c) mice were employed to study the role of GATA-1 signaling in in vitro IgE-mediated activation of bone marrow derived mast cells (BMMCs). Murine models of passive IgE-mediated and oral antigen-induced IgE-mediated anaphylaxis were employed in mice. Frequency of steady state mast cells in various tissues (duodenum, ear, and tongue), peritoneal cavity, and clinical symptoms (diarrhea, shock, and mast cell activation) and intestinal Type 2 immune cell analysis including CD4+ Th2 cells, type 2 innate lymphoid cells (ILC2), and IL-9 secreting mucosal mast cells (MMC9) were assessed. RESULTS: In vitro analysis revealed that ΔdblGata BMMCs exhibit a reduced maturation rate, decreased expression of FcεRIα, and degranulation capacity when compared to their wildtype (WT) counterparts. These in vitro differences did not impact tissue resident mast cell numbers, total IgE, and susceptibility to or severity of IgE-mediated passive anaphylaxis. Surprisingly, ΔdblGata mice were more susceptible to IgE-mast cell-mediated oral antigen induced anaphylaxis. The increased allergic response was associated with increased Type 2 immunity (antigen-specific IgE, and CD4+ TH2 cells), MMC9 cells and small intestine (SI) mast cell load. CONCLUSION: Diminished GATA-1 activity results in reduced in vitro mast cell FcεRIα expression, proliferation, and degranulation activity. However, in vivo, diminished GATA-1 activity results in normal homeostatic tissue mast cell levels and increased antigen-induced CD4+ Th2 and iMMC9 cell levels and heightened IgE-mast cell mediated reactions.
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Anafilaxia/etiologia , Hipersensibilidade Alimentar/etiologia , Fator de Transcrição GATA1/fisiologia , Imunoglobulina E/efeitos adversos , Mastócitos/imunologia , Deleção de Sequência , Índice de Gravidade de Doença , Anafilaxia/metabolismo , Anafilaxia/patologia , Animais , Hipersensibilidade Alimentar/metabolismo , Hipersensibilidade Alimentar/patologia , Imunoglobulina E/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos KnockoutRESUMO
Metastatic colonization involves paracrine/juxtacrine interactions with the microenvironment inducing an adaptive response through transcriptional regulation. However, the identities of transcription factors (TFs) induced by the metastatic microenvironment in ovarian cancer (OC) and their mechanism of action is poorly understood. Using an organotypic 3D culture model recapitulating the early events of metastasis, we identified ETS1 as the most upregulated member of the ETS family of TFs in metastasizing OC cells as they interacted with the microenvironment. ETS1 was regulated by p44/42 MAP kinase signaling activated in the OC cells interacting with mesothelial cells at the metastatic site. Human OC tumors had increased expression of ETS1, which predicted poor prognosis. ETS1 regulated OC metastasis both in vitro and in mouse xenografts. A combination of ChIP-seq and RNA-seq analysis and functional rescue experiments revealed FAK as the key transcriptional target and downstream effector of ETS1. Taken together, our results indicate that ETS1 is an essential transcription factor induced in OC cells by the microenvironment, which promotes metastatic colonization though the transcriptional upregulation of its target FAK.
Assuntos
Quinase 1 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Proteína Proto-Oncogênica c-ets-1/genética , Microambiente Tumoral/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Quinase 1 de Adesão Focal/metabolismo , Humanos , Estimativa de Kaplan-Meier , Camundongos Nus , Metástase Neoplásica , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína Proto-Oncogênica c-ets-1/metabolismo , Interferência de RNA , Transplante HeterólogoRESUMO
Sonic hedgehog (Shh) signaling influences neurogenesis and neural patterning during the development of central nervous system. Dysregulation of Shh signaling in brain leads to neurological disorders like autism spectrum disorder, depression, dementia, stroke, Parkinson's diseases, Huntington's disease, locomotor deficit, epilepsy, demyelinating disease, neuropathies as well as brain tumors. The synthesis, processing and transport of Shh ligand as well as the localization of its receptors and signal transduction in the central nervous system has been carefully reviewed. Further, we summarize the regulation of small molecule modulators of Shh pathway with potential in neurological disorders. In conclusion, further studies are warranted to demonstrate the potential of positive and negative regulators of the Shh pathway in neurological disorders.
Assuntos
Doenças do Sistema Nervoso , Transdução de Sinais , Transtorno do Espectro Autista , Proteínas Hedgehog , Humanos , NeurogêneseRESUMO
Food allergy is a harmful immune reaction driven by uncontrolled type 2 immune responses. Considerable evidence demonstrates the key roles of mast cells, IgE, and TH2 cytokines in mediating food allergy. However, this evidence provides limited insight into why only some, rather than all, food allergic individuals are prone to develop life-threatening anaphylaxis. Clinical observations suggest that patients sensitized to food through the skin early in life may later develop severe food allergies. Aberrant epidermal thymic stromal lymphopoietin and interleukin (IL) 33 production and genetic predisposition can initiate an allergic immune response mediated by dendritic cells and CD4+TH2 cells in inflamed skin. After allergic sensitization, intestinal IL-25 and food ingestion enhance concerted interactions between type 2 innate lymphoid cells (ILC2s) and CD4+TH2 cells, which perpetuate allergic reactions from the skin to the gut. IL-4 and cross-linking of antigen/IgE/FcεR complexes induce emigrated mast cell progenitors to develop into the multi-functional IL-9-producing mucosal mast cells, which produce prodigious amounts of IL-9 and mast cell mediators to drive intestinal mastocytosis in an autocrine loop. ILC2s and TH9 cells may also serve as alternative cellular sources of IL-9 to augment the amplification of intestinal mastocytosis, which is the key cellular checkpoint in developing systemic anaphylaxis. These findings provide a plausible view of how food allergy develops and progresses in a stepwise manner and that atopic signals, dietary allergen ingestion, and inflammatory cues are fundamental in promoting life-threatening anaphylaxis. This information will aid in improving diagnosis and developing more effective therapies for food allergy-triggered anaphylaxis.
Assuntos
Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/metabolismo , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Imunoglobulina E/imunologia , Interleucina-9/metabolismo , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Citocinas/metabolismo , Epiderme/imunologia , Epiderme/metabolismo , Alimentos/efeitos adversos , Hipersensibilidade Alimentar/patologia , Hipersensibilidade Alimentar/terapia , Humanos , Tolerância Imunológica/imunologia , Imunização , Interleucina-9/biossíntese , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Mastócitos/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
Genomic analysis of ovarian cancer cell lines has revealed a panel that best represents the most common ovarian cancer subtype, high-grade serous ovarian cancer (HGSOC). However, these HGSOC-like cell lines have not been extensively applied by ovarian cancer researchers to date, and the most commonly used cell lines in the ovarian cancer field do not genetically resemble the major clinical type of the disease. For the HGSOC-like lines to serve as suitable models, they need to be characterized for common functional assays. To achieve that objective, we systematically studied a panel of HGSOC cells CAOV3, COV362, Kuramochi, OVCAR4, OVCAR5, OVCAR8, OVSAHO and SNU119 for migration, invasion, proliferation, clonogenicity, EMT phenotype and cisplatin resistance. They exhibited a range of efficacies and OVCAR5, OVCAR8 and Kuramochi were the most aggressive. SNU119 and OVSAHO cells demonstrated the lowest functional activities. Wide differences in expression of EMT markers were observed between cell lines. SNU119 were the most epithelial and OVCAR8 had the most mesenchymal phenotype. COV362 was the most resistant to cisplatin while CAOV3 was the most sensitive. Taken together, our systematic characterization represents a valuable resource to help guide the application of HGSOC cells by the cancer research community.