RESUMO
Cytotoxic T lymphocytes (CTL) are pivotal in combating cancer, yet their efficacy is often hindered by the immunosuppressive tumor microenvironment, resulting in CTL exhaustion. This study investigates the role of interleukin-3 (IL3) in orchestrating antitumor immunity through CTL modulation. We found that intratumoral CTLs exhibited a progressive decline in IL3 production, which was correlated with impaired cytotoxic function. Augmenting IL3 supplementation, through intraperitoneal administration of recombinant IL3, IL3-expressing tumor cells, or IL3-engineered CD8+ T cells, conferred protection against tumor progression, concomitant with increased CTL activity. CTLs were critical for this therapeutic efficacy as IL3 demonstrated no impact on tumor growth in Rag1 knockout mice or following CD8+ T-cell depletion. Rather than acting directly, CTL-derived IL3 exerted its influence on basophils, concomitantly amplifying antitumor immunity within CTLs. Introducing IL3-activated basophils retarded tumor progression, whereas basophil depletion diminished the effectiveness of IL3 supplementation. Furthermore, IL3 prompted basophils to produce IL4, which subsequently elevated CTL IFNγ production and viability. Further, the importance of basophil-derived IL4 was evident from the absence of benefits of IL3 supplementation in IL4 knockout tumor-bearing mice. Overall, this research has unveiled a role for IL3-mediated CTL-basophil cross-talk in regulating antitumor immunity and suggests harnessing IL3 sustenance as a promising approach for optimizing and enhancing cancer immunotherapy. See related Spotlight, p. 798.
Assuntos
Interleucina-3 , Camundongos Knockout , Linfócitos T Citotóxicos , Animais , Camundongos , Linfócitos T Citotóxicos/imunologia , Interleucina-3/metabolismo , Interleucina-3/imunologia , Camundongos Endogâmicos C57BL , Linhagem Celular Tumoral , Microambiente Tumoral/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Comunicação Celular/imunologia , HumanosRESUMO
Interleukin-3 (IL-3) is a hematopoietic growth factor and critical regulator of inflammatory response such as sepsis. IL-3 binds to IL-3 receptor α (IL-3Rα), which is then associated with IL-3Rß to initiate signaling. How IL-3-triggered physiological and pathological effects are regulated at the receptor level is unclear. Here, we show that the plasma membrane-associated E3 ubiquitin ligase MARCH3 negatively regulates IL-3-triggered signaling. MARCH3 is associated with IL-3Rα, mediates its K48-linked polyubiquitination at K377 and promotes its proteasomal degradation. MARCH3-deficiency promotes IL-3-triggered transcription of downstream effector genes and IL-3-induced expansion of myeloid cells. In the cecal ligation and puncture (CLP) model of sepsis, MARCH3-deficiency aggravates IL-3-ampified expression of inflammatory cytokines, organ damage and inflammatory death. Our findings suggest that regulation of IL-3Rα by MARCH3 plays an important role in IL-3-triggered physiological functions and inflammatory diseases.
Assuntos
Subunidade alfa de Receptor de Interleucina-3/imunologia , Interleucina-3/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteólise , Ubiquitinação/imunologia , Animais , Células HEK293 , Humanos , Inflamação/genética , Inflamação/imunologia , Interleucina-3/genética , Subunidade alfa de Receptor de Interleucina-3/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Ubiquitinação/genéticaRESUMO
Regulatory T cells (Tregs) play a crucial role in modulating the inflammatory response and participated in sepsis-related immune dysfunctions. However, little is known about the regulatory mechanisms by which Tregs are kept in check during immune responses. Here, we verified the simultaneous expression of interleukin-3 (IL-3) and its receptor (IL-3R) in Tregs. Then, by modulation of IL-3 expression via lentiviral transduction-mediated small interfering RNA, we demonstrated that IL-3 negatively regulated Tregs activity via an autocrine mechanism. Furthermore, we found that anti-IL-3 antibody treatment significantly diminished inflammatory cytokines and organ injury, and improved survival in septic mice, which was associated with enhanced Treg percentage and function. Collectively, these results suggest that IL-3 negatively regulates the activity of Tregs in a previously unrecognized autocrine manner, and plays an important role in the excessive inflammatory response in sepsis, which might be utilized as a therapeutic strategy for the treatment of complications in sepsis.
Assuntos
Interleucina-3/imunologia , Sepse , Linfócitos T Reguladores , Animais , Citocinas , Camundongos , Sepse/imunologia , Sepse/fisiopatologia , Linfócitos T Reguladores/imunologiaRESUMO
Human basophils regulate allergic reactions by secreting histamine, interleukin 4 (IL-4) and IL-13 through key surface receptors FcεRI as well as IL-3R, which are constitutively expressed on basophils. IL-3/IL-3R signaling axis plays key roles in regulating the development and activation of basophils. We and others have shown that IL-3-induced surface receptors e.g. ST2, IL-17RB and IL-2 receptors regulate the biology of basophils. However, the expression and function of IL-3-induced surface proteins on human basophils remain to be elucidated. We in this study aimed to identify new basophil activation regulators by transcriptomic analysis of IL-3-stimulated basophils. Gene expression microarray analysis of IL-3-treated basophils revealed 2050 differentially expressed genes, of which 323 genes encoded surface proteins including GITR. We identified that GITR was preferentially induced by IL-3 rather than anti-IgE, IL-33, fMLP and C5a. IL-3-induced GITR was suppressed by inhibitors targeting JAK2, PI3K and MEK1/2. Stimulation of IL-3-treated basophils by GITR enhanced the expression of IL-4 and IL-13. Moreover, IgE-mediated degranulation was enhanced by GITRL in the presence of IL-3. This transcriptomic analysis of IL-3-activated basophils helps to identify novel activation regulator. IL-3-induced GITR promoted the activation of basophils, adding new evidence supporting GITR as an important player in Th2-associated immune responses.
Assuntos
Basófilos/imunologia , Regulação da Expressão Gênica/imunologia , Proteína Relacionada a TNFR Induzida por Glucocorticoide/imunologia , Interleucina-3/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Feminino , Humanos , MasculinoRESUMO
The clinical success of immune checkpoint modulators and the development of next-generation immune-oncology (IO) agents underscore the need for robust preclinical models to evaluate novel IO therapeutics. Human immune system (HIS) mouse models enable in vivo studies in the context of the HIS via a human tumor. The immunodeficient mouse strains NOG (Prkdcscid Il2rgtm1Sug ) and triple-transgenic NOG-EXL [Prkdcscid Il2rgtm1Sug Tg (SV40/HTLV-IL3, CSF2)], which expresses human IL-3 and GM-CSF, allow for human CD34+ hematopoietic stem cell (huCD34+ HSC) engraftment and multilineage immune cell development by 12 to 16 weeks post-transplant and facilitate studies of immunomodulatory agents. A more rapid model of human immune engraftment utilizes peripheral blood mononuclear cells (PBMCs) transplanted into immunodeficient murine hosts, permitting T-cell engraftment within 2 to 3 weeks without outgrowth of other human immune cells. The PBMC-HIS model can be limited due to onset of xenogeneic graft-versus-host disease (xGVHD) within 3 to 5 weeks post-implantation. Host deficiency in MHC class I, as occurs in beta-2 microglobulin knockout in either NOG or NSG mice, results in resistance to xGVHD, which permits a longer therapeutic window. In this article, detailed processes for generating humanized mice by transplantation of HSCs from cord blood-derived huCD34+ HSCs or PBMCs into immunodeficient mouse strains to respectively generate HSC-HIS and PBMC-HIS mouse models are provided. In addition, the co-engraftment and growth kinetics of patient-derived and cell line-derived xenograft tumors in humanized mice and recovery of tumor-infiltrating lymphocytes from growing tumors to evaluate immune cell subsets by flow cytometry are described. © 2020 The Authors. Basic Protocol 1: Establishment of patient-derived xenograft tumors in CD34+ hematopoietic stem cell-humanized mice Basic Protocol 2: Establishment of patient-derived xenograft tumors in peripheral blood mononuclear cell-humanized mice Support Protocol 1: Flow cytometry assessment of humanization in mice Support Protocol 2: Flow cytometry assessment of tumor-infiltrating lymphocytes in tumor-bearing humanized mouse models.
Assuntos
Antígenos CD34/imunologia , Sangue Fetal/imunologia , Células-Tronco Hematopoéticas/imunologia , Leucócitos Mononucleares/imunologia , Animais , Modelos Animais de Doenças , Feminino , Doença Enxerto-Hospedeiro/imunologia , Fatores Imunológicos/imunologia , Subunidade gama Comum de Receptores de Interleucina/imunologia , Interleucina-3/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T/imunologiaRESUMO
IL-3, a cytokine secreted by activated T lymphocytes, is known to regulate the proliferation, survival, and differentiation of hematopoietic cells. However, the role of IL-3 in regulation of T cell functions is not fully delineated. Previously, we have reported that IL-3 plays an important role in development of regulatory T cells in mice. In this study, we investigated the regulation of IL-3R expression on human Th cells and also examined the role of IL-3 in effector functions of these cells. We found that human peripheral blood Th cells in resting state do not show surface expression of IL-3R; however, its expression was observed at transcript and intracellular protein levels. The functional IL-3R expression on the surface was seen only after antigenic stimulation. When naive Th cells were activated in the presence of various cytokines, we found that IL-4 significantly increases the surface expression of IL-3R and also increases the number of IL-3R+ Th cells. Interestingly, IL-3R+ cells exhibit a Th2 cell-like phenotype and show high GATA-3 expression. Moreover, Th2 cells in presence of IL-3 show increased expression of type 2 effector cytokines, such as IL-4, IL-5, and IL-13. Furthermore, IL-3R expressing and IL-3-secreting Th cells were high in house dust mite-allergic patients. Thus, to our knowledge, we provide the first evidence that the expression of IL-3R on activated human Th cells is modulated by IL-4, and IL-3 regulates the effector functions of Th2 cells. Our results suggest that IL-3 may play an important role in regulating allergic immune responses.
Assuntos
Diferenciação Celular/imunologia , Interleucina-3/imunologia , Interleucina-4/imunologia , Receptores de Interleucina-3/imunologia , Células Th2/imunologia , Humanos , Hipersensibilidade/imunologia , Interleucina-3/metabolismo , Interleucina-4/metabolismo , Ativação Linfocitária/imunologia , Receptores de Interleucina-3/metabolismoRESUMO
BACKGROUND: The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Cell-free eosinophil granules are found in tissues in eosinophilic diseases, including asthma. This suggests that eosinophils have lysed and released cellular content, likely harming tissues. OBJECTIVE: The present study explores the mechanism of CD32- and αMß2 integrin-dependent eosinophil cytolysis of IL3-primed blood eosinophils seeded on heat-aggregated immunoglobulin G (HA-IgG). METHODS: Cytoskeletal events and signalling pathways potentially involved in cytolysis were assessed using inhibitors. The level of activation of the identified events and pathways involved in cytolysis was measured. In addition, the links between these identified pathways and changes in degranulation (exocytosis) and adhesion were analysed. RESULTS: Cytolysis of IL3-primed eosinophils was dependent on the production of reactive oxygen species (ROS) and downstream phosphorylation of p-38 MAPK. In addition, formation of microtubule (MT) arrays was necessary for cytolysis and was accompanied by changes in MT dynamics as measured by phosphorylation status of stathmin and microtubule-associated protein 4 (MAP4), the latter of which was regulated by ROS production. Reduced ROCK signalling preceded cytolysis, which was associated with eosinophil adhesion and reduced migration. CONCLUSION AND CLINICAL RELEVANCE: In this CD32- and αMß2 integrin-dependent adhesion model, lysing eosinophils exhibit reduced migration and ROCK signalling, as well as both MT dynamic changes and p-38 phosphorylation downstream of ROS production. We propose that interfering with these pathways would modulate eosinophil cytolysis and subsequent eosinophil-driven tissue damage.
Assuntos
Eosinófilos/imunologia , Imunoglobulina G/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Microtúbulos/imunologia , Humanos , Interleucina-3/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Espécies Reativas de Oxigênio/imunologia , Receptores de IgG/imunologia , Quinases Associadas a rhoRESUMO
Helicobacter pylori infection occurs throughout the world between countries and races. A seroprevalence study of H. pylori infection in the occurrence of atherosclerosis (ASc) has been considered to be the main cause of cardiovascular diseases (CVDs) in part of Iran. From 600 volunteers, 97 serum samples were selected and tested for the presence of H. pylori infection. Positive and negative H. pylori infection samples were then selected for Chlamydia pneumonia and cytomegalovirus infection. Their serum levels for high-sensitive C-reactive protein (hs-CRP) and IL-6 titer were measured using an ELISA test. Prevalence of H. pylori infection was â¼68.04% in Karaj. Among the 22 samples of cytokine titration, 4 individuals were HP- and 18 were HP+. It was also noticed that 17 samples had normal titration (77.27%) of IL-6 and 3 had a high level of IL-6 (13.63%), 20 people had high hs-CRP titration (90.90%), and 2 were low level hs-CRP (9.1%). Obtained results showed that there is no significant difference between HP+ and HP- infection and IL-6 and hs-CRP titration. Results showed that H. pylori could not lead to ASc by increasing the levels of proinflammatory cytokines such as IL-6 and hs-CRP. Further studies are necessary for the detection of mechanism of H. pylori on ASc.
Assuntos
Aterosclerose/sangue , Doenças Cardiovasculares/sangue , Infecções por Helicobacter/sangue , Helicobacter pylori/imunologia , Adolescente , Adulto , Idoso , Anticorpos Antibacterianos/sangue , Aterosclerose/epidemiologia , Aterosclerose/imunologia , Proteína C-Reativa/metabolismo , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/microbiologia , Criança , Feminino , Infecções por Helicobacter/epidemiologia , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Humanos , Interleucina-3/imunologia , Interleucina-6/imunologia , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Estudos Soroepidemiológicos , Adulto JovemRESUMO
Despite recent advances in asthma management with anti-IL-5 therapies, many patients have eosinophilic asthma that remains poorly controlled. IL-3 shares a common ß subunit receptor with both IL-5 and GM-CSF but, through α-subunit-specific properties, uniquely influences eosinophil biology and may serve as a potential therapeutic target. We aimed to globally characterize the transcriptomic profiles of GM-CSF, IL-3, and IL-5 stimulation on human circulating eosinophils and identify differences in gene expression using advanced statistical modeling. Human eosinophils were isolated from the peripheral blood of healthy volunteers and stimulated with either GM-CSF, IL-3, or IL-5 for 48 h. RNA was then extracted and bulk sequencing performed. DESeq analysis identified differentially expressed genes and weighted gene coexpression network analysis independently defined modules of genes that are highly coexpressed. GM-CSF, IL-3, and IL-5 commonly upregulated 252 genes and downregulated 553 genes, producing a proinflammatory and survival phenotype that was predominantly mediated through TWEAK signaling. IL-3 stimulation yielded the most numbers of differentially expressed genes that were also highly coexpressed (n = 119). These genes were enriched in pathways involving JAK/STAT signaling. GM-CSF and IL-5 stimulation demonstrated redundancy in eosinophil gene expression. In conclusion, IL-3 produces a distinct eosinophil gene expression program among the ß-chain receptor cytokines. IL-3-upregulated genes may provide a foundation for research into therapeutics for patients with eosinophilic asthma who do not respond to anti-IL-5 therapies.
Assuntos
Citocinas/imunologia , Eosinófilos/imunologia , Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Interleucina-3/imunologia , Interleucina-5/imunologia , Asma/imunologia , Regulação para Baixo/imunologia , Humanos , Transdução de Sinais/imunologia , Regulação para Cima/imunologiaRESUMO
Basophils are a small population of innate immune cells, but their release of the cytokine interleukin-4 (IL-4) is important for mounting an efficient immune response against distinct parasites. Yoshikawa et al (in the 9 April 2019 issue) showed that whereas STIM1 is essential for IL-4 release after stimulation of FcεRI, STIM2 mediates a delayed IL-3/IL-33-induced IL-4 release independent of STIM1.
Assuntos
Interleucina-3/imunologia , Doenças Parasitárias/imunologia , Molécula 2 de Interação Estromal/imunologia , Animais , Humanos , Interleucina-33/imunologia , Interleucina-4/imunologia , Proteínas de Neoplasias/imunologia , Doenças Parasitárias/patologia , Receptores de IgE/imunologia , Molécula 1 de Interação Estromal/imunologiaRESUMO
Basophils have nonredundant roles in various immune responses that require Ca2+ influx. Here, we examined the role of two Ca2+ sensors, stromal interaction molecule 1 and 2 (STIM1 and STIM2), in basophil activation. We found that loss of STIM1, but not STIM2, impaired basophil IL-4 production after stimulation with immunoglobulin E (IgE)-containing immune complexes. In contrast, when basophils were stimulated with IL-3, loss of STIM2, but not STIM1, reduced basophil IL-4 production. This difference in STIM proteins was associated with distinct time courses of Ca2+ influx and transcription of the Il4 gene that were elicited by each stimulus. Similarly, basophil-specific STIM1 expression was required for IgE-driven chronic allergic inflammation in vivo, whereas STIM2 was required for IL-4 production after combined IL-3 and IL-33 treatment in mice. These data indicate that STIM1 and STIM2 have differential roles in the production of IL-4, which are stimulus dependent. Furthermore, these results illustrate the vital role of STIM2 in basophils, which is often considered to be less important than STIM1.
Assuntos
Basófilos/imunologia , Sinalização do Cálcio/imunologia , Interleucina-3/imunologia , Interleucina-4/imunologia , Molécula 1 de Interação Estromal/imunologia , Molécula 2 de Interação Estromal/imunologia , Animais , Basófilos/citologia , Sinalização do Cálcio/genética , Imunoglobulina E/imunologia , Interleucina-3/genética , Interleucina-4/genética , Camundongos , Camundongos Knockout , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genéticaRESUMO
One successful class of cancer immunotherapies, immune checkpoint inhibitory antibodies, disrupts key pathways that regulate immune checkpoints, such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). These agents unleash the potency of antigen-experienced T cells that have already been induced as a consequence of the existing tumor. But only 20% of cancers naturally induce T cells. For most cancers, vaccines are require to induce and mobilize T effector cells (Teffs ) to traffick into tumors. We evaluated the effects of anti-CTLA-4 given in combination with an antigen-specific dendritic cell vaccine on intratumoral Teffs in a murine pancreatic cancer model. The dendritic cell-targeted tumor antigen plus anti-CTLA-4 significantly increased the number of vaccine-induced CD4+ Teffs within the tumor. This increase was accompanied by a reduction in the size of the peripheral CD4+ Teff pool. We also found that IL-3 production by activated CD4+ T cells was significantly increased with this combination. Importantly, the CD4+ Teff response was attenuated in Il3-/- mice, suggesting mediation of the effect by IL-3. Finally, the induced T cell infiltration was associated with activation of the tumor endothelium by T cell-derived IL-3. Our findings collectively provide a new insight into the mechanism driving Teff infiltration and vascular activation in a murine pancreatic cancer model, specifically identifying a new role for IL-3 in the anticancer immune response.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Antígeno CTLA-4/antagonistas & inibidores , Vacinas Anticâncer/imunologia , Interleucina-3/imunologia , Neoplasias Pancreáticas/patologia , Animais , Linfócitos T CD4-Positivos/imunologia , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Modelos Animais de Doenças , Interleucina-3/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Pancreáticas/imunologiaRESUMO
The ß common chain cytokines GM-CSF, IL-3, and IL-5 regulate varied inflammatory responses that promote the rapid clearance of pathogens but also contribute to pathology in chronic inflammation. Therapeutic interventions manipulating these cytokines are approved for use in some cancers as well as allergic and autoimmune disease, and others show promising early clinical activity. These approaches are based on our understanding of the inflammatory roles of these cytokines; however, GM-CSF also participates in the resolution of inflammation, and IL-3 and IL-5 may also have such properties. Here, we review the functions of the ß common cytokines in health and disease. We discuss preclinical and clinical data, highlighting the potential inherent in targeting these cytokine pathways, the limitations, and the important gaps in understanding of the basic biology of this cytokine family.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Inflamação/imunologia , Interleucina-3/imunologia , Interleucina-5/imunologia , Animais , Doenças Autoimunes/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/deficiência , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Hematopoese/imunologia , Humanos , Inflamação/terapia , Interleucina-3/antagonistas & inibidores , Interleucina-3/deficiência , Interleucina-3/genética , Interleucina-5/antagonistas & inibidores , Interleucina-5/deficiência , Interleucina-5/genética , Camundongos , Camundongos Knockout , Família Multigênica , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/imunologia , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/imunologia , Receptores de Interleucina-5/genética , Receptores de Interleucina-5/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais , Relação Estrutura-Atividade , Vacinação , Cicatrização/imunologiaRESUMO
CD8+ T cell immunosurveillance is crucial in solid tumors and T cell dysfunction leads to tumor progression. In contrast, the role of CD8+ T cells in the control of leukemia is less clear. We characterized the molecular signature of leukemia stem/progenitor cells (LSPCs) and paired CD8+ T cells in patients with acute myeloid leukemia (AML). Epigenetic alterations via histone deacetylation reduced the expression of immune-related genes in bone marrow (BM)-infiltrating CD8+ T cells. Surprisingly, a silenced gene expression pattern in CD8+ T cells significantly correlated with an improved prognosis. To define interactions between CD8+ T cells and LSPCs, we performed comprehensive correlative network modeling. This analysis indicated that CD8+ T cells contribute to the maintenance/expansion of LSPCs, particularly in favorable risk AML. Functionally, CD8+ T cells in favorable AML induced the expansion of LSPCs by stimulating the autocrine production of important hematopoietic cytokines such as interleukin (IL)-3. In contrast, LSPCs in aggressive AML were characterized by a higher activation of stemness/proliferation-related pathways and develop independent of BM CD8+ T cells. Overall, our study indicates that CD8+ T cells support and expand LSPCs in favorable risk AML whereas intermediate and adverse risk AML possess the intrinsic molecular abnormalities to develop independently.
Assuntos
Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Leucemia Mieloide Aguda/imunologia , Células-Tronco/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células/fisiologia , Citocinas/imunologia , Humanos , Interleucina-3/imunologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Adulto JovemRESUMO
Acquisition of self-reactive effector CD4+ T cells is a major component of the autoimmune response that can occur during myocarditis, an inflammatory form of cardiomyopathy. Although the processes by which self-reactive T cells gain effector function have received considerable attention, how these T cells contribute to effector organ inflammation and damage is less clear. Here, we identified an IL-3-dependent amplification loop that exacerbates autoimmune inflammation. In experimental myocarditis, we show that effector organ-accumulating autoreactive IL-3+ CD4+ T cells stimulate IL-3R+ tissue macrophages to produce monocyte-attracting chemokines. The newly recruited monocytes differentiate into antigen-presenting cells that stimulate local IL-3+ CD4+ T cell proliferation, thereby amplifying organ inflammation. Consequently, Il3 -/- mice resist developing robust autoimmune inflammation and myocardial dysfunction, whereas therapeutic IL-3 targeting ameliorates disease. This study defines a mechanism that orchestrates inflammation in myocarditis, describes a previously unknown function for IL-3, and identifies IL-3 as a potential therapeutic target in patients with myocarditis.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Quimiotaxia/imunologia , Interleucina-3/imunologia , Monócitos/imunologia , Miocardite/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Linfócitos T CD4-Positivos/patologia , Proliferação de Células , Quimiotaxia/genética , Interleucina-3/genética , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/patologia , Miocardite/genética , Miocardite/patologiaRESUMO
A large number of animal models revealed that IL-3 plays an important role for the development of T and B cell-mediated autoimmune diseases. However, little is known about the expression and regulation of IL-3 receptors in human T and B cells and how IL-3 modulates the activation and survival of these cells. We show that the IL-3 receptor CD123 is substantially upregulated on proliferating CD4+ and CD8+ T as well as B cells. Upregulation of CD123 differs between various activators and can be further modulated by cytokines. Exposure of human T and B cells to IL-3 enhances proliferation and survival. IL-3 also induces a shift towards secretion of proinflammatory cytokines in T and B cells and reduces the expression of IL-10 in B cells. Thus IL-3 may have proinflammatory and immunostimulatory properties also in human autoimmune diseases.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Interleucina-3/imunologia , Receptores de Interleucina-3/imunologia , Doenças Autoimunes/imunologia , Citocinas/imunologia , Humanos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Ativação Linfocitária/imunologia , Regulação para Cima/imunologiaRESUMO
Periostin, which is induced by interleukin (IL)-13, is an extracellular matrix (ECM) protein that supports αMß2 integrin-mediated adhesion and migration of IL-5-stimulated eosinophils. Transforming growth factor (TGF)-ß-induced protein (TGFBI) is a widely expressed periostin paralog known to support monocyte adhesion. Our objective was to compare eosinophil adhesion and migration on TGFBI and periostin in the presence of IL-5-family cytokines. Eosinophil adhesion after 1 h and random motility over 20 h in the presence of various concentrations of IL-5, IL-3, or granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified in wells coated with various concentrations of TGFBI or periostin. Results were compared to video microscopy of eosinophils. Cytokine-stimulated eosinophils adhered equivalently well to TGFBI or periostin in a coating concentration-dependent manner. Adhesion was blocked by anti-αMß2 and stimulated at the lowest concentration by GM-CSF. In the motility assay, periostin was more potent than TGFBI, the coating-concentration effect was bimodal, and IL-3 was the most potent cytokine. Video microscopy revealed that under the optimal coating condition of 5 µg/ml periostin, most eosinophils migrated persistently and were polarized and acorn-shaped with a ruffling forward edge and granules gathered together, in front of the nucleus. On 10 µg/ml periostin or TGFBI, more eosinophils adopted a flattened pancake morphology with dispersed granules and nuclear lobes, and slower migration. Conversion between acorn and pancake morphologies were observed. We conclude that TGFBI or periostin supports two modes of migration by IL-5 family cytokine-activated eosinophils. The rapid mode is favored by intermediate protein coatings and the slower by higher coating concentrations. We speculate that eosinophils move by haptotaxis up a gradient of adhesive ECM protein and then slow down to surveil the tissue.
Assuntos
Moléculas de Adesão Celular/imunologia , Eosinófilos/citologia , Proteínas da Matriz Extracelular/imunologia , Fator de Crescimento Transformador beta/imunologia , Adesão Celular , Ensaios de Migração de Leucócitos , Movimento Celular , Eosinófilos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Interleucina-3/imunologia , Interleucina-5/imunologiaRESUMO
Binding of allergen-specific IgE to its primary receptor FcεRI on basophils and mast cells represents a central event in the development of allergic diseases. The high-affinity interaction between IgE and FcεRI results in permanent sensitization of these allergic effector cells and critically regulates their release of pro-inflammatory mediators upon IgE cross-linking by allergens. In addition, binding of monomeric IgE has been reported to actively regulate FcεRI surface levels and promote survival of mast cells in the absence of allergen through the induction of autocrine cytokine secretion including interleukin-3 (IL-3). As basophils and mast cells share many biological commonalities we sought to assess the role of monomeric IgE binding and IL-3 signaling in FcεRI regulation and cell survival of primary human basophils. FcεRI cell surface levels and survival of isolated blood basophils were assessed upon addition of monomeric IgE or physiologic removal of endogenous cell-bound IgE with a disruptive IgE inhibitor by flow cytometry. We further determined basophil cell numbers in both low and high serum IgE blood donors and mice that are either sufficient or deficient for FcεRI. Ultimately, we investigated the effect of IL-3 on basophil surface FcεRI levels by protein and gene expression analysis. Surface levels of FcεRI were passively stabilized but not actively upregulated in the presence of monomeric IgE. In contrast to previous observations with mast cells, monomeric IgE binding did not enhance basophil survival. Interestingly, we found that IL-3 transcriptionally regulates surface levels of FcεRI in human primary basophils. Our data suggest that IL-3 but not monomeric IgE regulates FcεRI expression and cell survival in primary human basophils. Thus, blocking of IL-3 signaling in allergic effector cells might represent an interesting approach to diminish surface FcεRI levels and to prevent prolonged cell survival in allergic inflammation.
Assuntos
Basófilos/imunologia , Hipersensibilidade/genética , Imunoglobulina E/genética , Interleucina-3/genética , Receptores de IgE/genética , Animais , Basófilos/efeitos dos fármacos , Basófilos/patologia , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/fisiopatologia , Imunoglobulina E/imunologia , Interleucina-3/imunologia , Interleucina-3/farmacologia , Interleucina-5/genética , Interleucina-5/imunologia , Interleucina-5/farmacologia , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Receptores de IgE/deficiência , Receptores de IgE/imunologia , Transdução de Sinais , Transcrição GênicaRESUMO
BACKGROUND: Basophils are commonly associated with allergic responses because of their ability to produce large amounts of pro-Th2 cytokines and histamine. However, the mechanisms through which bone marrow-resident basophils (BMRB) become fully competent cytokine and histamine producers in response to IgE crosslinking are poorly understood. Here, we sought to determine the role of IL-3 in promoting pro-Th2 basophils. METHODS: BMRB and basophils exposed to IL-3 in vitro and in vivo were evaluated for their production of Th2 cytokines and histamine in response to FcεRI crosslinking on both protein and gene expression levels. In vivo relevance of our findings was assessed in a model of ovalbumin-induced allergic asthma using IL-3-deficient and wild-type mice in a protocol of adoptive basophil transfer. RESULTS: We show that BMRB and basophils previously exposed to IL-3 differ in their ability to generate cytokines (IL-4, IL-6, IL-13, and GM-CSF) and histamine in response to FcεRI crosslinking, reflecting two stages of maturation. Exposure to IL-3 initiated an autocrine loop of endogenous IL-3 production that enhanced histamine and cytokine production upon FcεRI crosslinking. This increased responsiveness required calcium flux and was dependent on calcineurin and store-operated calcium channels. Our findings are of pathophysiological relevance, as assessed by the failure of IL-3-deficient mice to develop airway hyperreactivity, which could be restored by adoptive transfer of IL-3-derived basophils recovered from wild-type mice. CONCLUSION: IL-3-dependent basophils promote Th2 allergic AHR, which designates the IL-3/basophil axis as a promising therapeutic target for the treatment of basophil-dependent asthma.
Assuntos
Interleucina-3/imunologia , Hipersensibilidade Respiratória/etiologia , Animais , Basófilos , Células da Medula Óssea , Citocinas/metabolismo , Histamina/metabolismo , Inflamação/patologia , Camundongos , Hipersensibilidade Respiratória/patologia , Células Th2/imunologia , Células Th2/fisiologiaRESUMO
Early acting cytokines and growth factors such as those of the CD131 ßc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.