RESUMO
Elevated concentrations of tumor necrosis factor-α (TNF-α) are detected in pathologies characterized by chronic inflammation. Whether TNF-α plays a role in manipulating the host's immune system toward generating an immunosuppressive milieu, typical of ongoing chronic inflammation, is unclear. Here we showed that TNF-α exhibited a dual function during chronic inflammation: arresting differentiation of immature myeloid-derived suppressor cells (MDSCs) primarily via the S100A8 and S100A9 inflammatory proteins and their corresponding receptor (RAGE) and augmenting MDSC suppressive activity. These functions led to in vivo T and NK cell dysfunction accompanied by T cell antigen receptor ζ chain downregulation. Furthermore, administration of etanercept (TNF-α antagonist) during early chronic inflammatory stages reduced MDSCs' suppressive activity and enhanced their maturation into dendritic cells and macrophages, resulting in the restoration of in vivo immune functions and recovery of ζ chain expression. Thus, TNF has a fundamental role in promoting an immunosuppressive environment generated during chronic inflammation.
Assuntos
Diferenciação Celular/imunologia , Inflamação/imunologia , Células Mieloides/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Calgranulina A/genética , Calgranulina A/imunologia , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/imunologia , Calgranulina B/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Doença Crônica , Etanercepte , Citometria de Fluxo , Expressão Gênica/imunologia , Immunoblotting , Imunoglobulina G/farmacologia , Inflamação/genética , Inflamação/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores do Fator de Necrose Tumoral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genéticaRESUMO
The mucosal immune system plays a vital role in protecting the host from the external environment. Its major challenge is to balance immune responses against harmful and harmless agents and serve as a 'homeostatic gate keeper'. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of undifferentiated cells that are characterized by an immunoregulatory and immunosuppressive phenotype. Herein we postulate that MDSCs may be involved in shaping immune responses related to mucosal immunity, due to their immunomodulatory and tissue remodeling functions. Until recently, MDSCs were investigated mainly in cancerous diseases, where they induce and contribute to an immunosuppressive and inflammatory environment that favors tumor development. However, it is now becoming clear that MDSCs participate in non-cancerous conditions such as chronic infections, autoimmune diseases, pregnancy, aging processes and immune tolerance to commensal microbiota at mucosal sites. Since MDSCs are found in the periphery only in small numbers under normal conditions, their role is highlighted during pathologies characterized by acute or chronic inflammation, when they accumulate and become activated. In this review, we describe several aspects of the current knowledge characterizing MDSCs and their involvement in the regulation of the mucosal epithelial barrier, their crosstalk with commensal microbiota and pathogenic microorganisms, and their complex interactions with a variety of surrounding regulatory and effector immune cells. Finally, we discuss the beneficial and harmful outcomes of the MDSC regulatory functions in diseases affecting mucosal tissues. We wish to illuminate the pivotal role of MDSCs in mucosal immunity, the limitations in our understanding of all the players and the intricate challenges stemming from the complex interactions of MDSCs with their environment.
Assuntos
Epitélio/imunologia , Inflamação/imunologia , Microbiota/imunologia , Células Supressoras Mieloides/imunologia , Animais , Homeostase , Interações Hospedeiro-Parasita , Humanos , Imunidade nas Mucosas , ImunomodulaçãoRESUMO
Chronic inflammation is associated with immunosuppression and downregulated expression of the TCR CD247. In searching for new biomarkers that could validate the impaired host immune status under chronic inflammatory conditions, we discovered that sorting nexin 9 (SNX9), a protein that participates in early stages of clathrin-mediated endocytosis, is downregulated as well under such conditions. SNX9 expression was affected earlier than CD247 by the generated harmful environment, suggesting that it is a potential marker sensing the generated immunosuppressive condition. We found that myeloid-derived suppressor cells, which are elevated in the course of chronic inflammation, are responsible for the observed SNX9 reduced expression. Moreover, SNX9 downregulation is reversible, as its expression levels return to normal and immune functions are restored when the inflammatory response and/or myeloid-derived suppressor cells are neutralized. SNX9 downregulation was detected in numerous mouse models for pathologies characterized by chronic inflammation such as chronic infection (Leishmania donovani), cancer (melanoma and colorectal carcinoma), and an autoimmune disease (rheumatoid arthritis). Interestingly, reduced levels of SNX9 were also observed in blood samples from colorectal cancer patients, emphasizing the feasibility of its use as a diagnostic and prognostic biomarker sensing the host's immune status and inflammatory stage. Our new discovery of SNX9 as being regulated by chronic inflammation and its association with immunosuppression, in addition to the CD247 regulation under such conditions, show the global impact of chronic inflammation and the generated immune environment on different cellular pathways in a diverse spectrum of diseases.
Assuntos
Complexo CD3/biossíntese , Hospedeiro Imunocomprometido/imunologia , Inflamação/imunologia , Células Mieloides/imunologia , Nexinas de Classificação/biossíntese , Animais , Artrite Reumatoide/imunologia , Biomarcadores Tumorais/sangue , Proliferação de Células , Células Cultivadas , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/patologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Masculino , Melanoma/diagnóstico , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Nexinas de Classificação/sangue , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Chronic inflammation arising in a diverse range of non-cancerous and cancerous diseases, dysregulates immunity and exposes patients to a variety of complications. These include immunosuppression, tissue damage, cardiovascular diseases and more. In cancer, chronic inflammation and related immunosuppression can directly support tumor growth and dramatically reduce the efficacies of traditional treatments, as well as novel immune-based therapies, which require a functional immune system. Nowadays, none of the immune biomarkers, regularly used by clinicians can sense a developing chronic inflammation, thus complications can only be detected upon their appearance. This review focuses on the necessity for such immune status biomarkers, which could predict complications prior to their appearance. Herein we bring examples for the use of cellular and molecular biomarkers in diagnosis, prognosis and follow-up of patients suffering from various cancers, for prediction of response to immune-based anti-cancer therapy and for prediction of cardiovascular disease in type 2 diabetes patients. Monitoring such biomarkers is expected to have a major clinical impact in addition to unraveling of the entangled complexity underlying dysregulated immunity in chronic inflammation. Thus, newly discovered biomarkers and those that are under investigation are projected to open a new era towards combating the silent damage induced by chronic inflammation.
Assuntos
Biomarcadores/metabolismo , Sistema Imunitário , Mediadores da Inflamação/metabolismo , Inflamação/imunologia , Neoplasias/imunologia , Animais , Detecção Precoce de Câncer , Humanos , Tolerância Imunológica , Testes Imunológicos , Inflamação/diagnóstico , Neoplasias/diagnósticoRESUMO
The inflamed tumor microenvironment plays a critical role in tumorigenesis. However, the mechanisms through which immune cells, particularly macrophages, promote tumorigenesis have only been partially elucidated, and the full scope of signaling pathways supplying macrophages with protumorigenic phenotypes still remain largely unknown. Here we report that germ-line absence of c-Jun N-terminal phosphorylation at serines 63 and 73 impedes inflammation-associated hepatocarcinogenesis, yet deleting c-Jun only in hepatocytes does not inhibit hepatocellular carcinoma (HCC) formation. Moreover, in human HCC-bearing livers, c-Jun phosphorylation is found in inflammatory cells, whereas it is mostly absent from malignant hepatocytes. Interestingly, macrophages in livers of mice with chronic hepatitis gradually switch their phenotype along the course of disease. Macrophage phenotype and density are dictated by c-Jun phosphorylation, in vitro and in vivo. Transition of macrophage phenotype, from antitumorigenic to protumorigenic, occurs before tumorigenesis, resulting in the production of various chemokines, including chemokine (C-C motif) ligand 17 (CCL17) and CCL22. Such signals, emanating from the liver microenvironment, direct the recruitment of regulatory T cells, which are known to facilitate HCC growth. Our findings identify c-Jun phosphorylation as a key mediator of macrophage education and point to the recruitment of immunosuppressive regulatory T cells as a possible protumorigenic mechanism.
Assuntos
Macrófagos/citologia , Macrófagos/imunologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Apoptose , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Quimiocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Hepatite/metabolismo , Hepatócitos/citologia , Humanos , Imunidade Inata , Inflamação , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Fosforilação , Prognóstico , Estrutura Terciária de Proteína , Microambiente TumoralRESUMO
Chronic inflammation, typical of various diseases including cancer, is a "silent bomb within the body," leading to complications that are only evident in most cases upon their appearance, when disease is already deteriorated. Chronic inflammation is associated with accumulation of myeloid-derived suppressor cells (MDSCs), which lead to immunosuppression. MDSCs have numerous harmful effects as they support tumor initiation, tumor growth and spreading, which in turn, perpetuate the inflammatory and suppressive conditions, thus preventing anticancer responses. As the concept of the immune system combating many types of tumors was revived in recent years, immunotherapy has dramatically changed the view of cancer treatment, and numerous novel therapies have been developed and approved by the FDA. However, cumulative clinical data point at very limited success rates. It is most likely that the developing chronic inflammation and MDSC-induced immunosuppression interfere with responses to such treatments and hence are major obstacles in achieving higher response rates to immune-based therapies. Moreover, chemotherapies were shown to have adverse immunoregulatory effects, enhancing or decreasing MDSC levels and activity, thus affecting treatment success. Therefore, therapeutic manipulations of chronic inflammation and MDSCs during cancer development are likely to enhance efficacy of immune- and chemo-based treatments, switching chronic pro-cancer inflammatory environments to an anticancerous milieu. Based on the functional relevance of immune networking in tumors, it is critical to merge monitoring immune system biomarkers into the traditional patient's categorization and treatment regimens. This will provide new tools for clinical practice, allowing appropriate management of cancer patients toward a better-personalized medicine.
Assuntos
Imunoterapia/métodos , Células Supressoras Mieloides/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Humanos , Células Supressoras Mieloides/patologia , Neoplasias/patologia , Microambiente Tumoral/imunologiaRESUMO
TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance.
Assuntos
Citoesqueleto/metabolismo , Sinapses Imunológicas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Actinas/metabolismo , Motivos de Aminoácidos/genética , Animais , Células Cultivadas , Citocinas/metabolismo , Feminino , Sinapses Imunológicas/imunologia , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Mutantes , Mutação/genética , Agregação de Receptores/genética , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The T-cell receptor (TCR) functions in both antigen recognition and signal transduction, which are crucial initial steps of antigen-specific immune responses. TCR integrity is vital for the induction of optimal and efficient immune responses, including the routine elimination of invading pathogens and the elimination of modified cells and molecules. Of the TCR subunits, the zeta-chain has a key role in receptor assembly, expression and signalling. Downregulation of TCR zeta-chain expression and impairment of T-cell function have been shown for T cells isolated from hosts with various chronic pathologies, including cancer, and autoimmune and infectious diseases. This review summarizes studies of the various pathologies that show this phenomenon and provides new insights into the mechanism responsible for downregulation of zeta-chain expression, its relevance and its clinical implications.
Assuntos
Imunidade/fisiologia , Inflamação/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Animais , Doenças Transmissíveis/imunologia , Regulação para Baixo , Humanos , Imunidade/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/fisiologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Chronic inflammation typical to various chronic diseases is associated with immunosuppression, mediated primarily by immature myeloid-derived suppressor cells (MDSCs). A variety of factors induce MDSC differentiation arrest, thus manipulating the host's immune function and suppressing the innate and adaptive immune systems, as reflected by their impaired status associated with down-regulated expression of the CD247 molecule. Such chronic inflammation-induced immunosuppressive features are also found in many tumors, generating tumor micro- and macro-environments that act as critical barriers to effective anti-tumor responses and therapies. This knowledge offers new and novel candidate immune targets for therapeutic interventions, in combination with more conventional approaches as chemotherapy, radiotherapy, and cancer cell targeted therapy. Therapeutic manipulation of chronic inflammation during cancer development is likely to enhance efficacy of treatments such as vaccinations, and adoptive T cell transfer, thus switching the chronic pro-cancer inflammatory environments into an anti-cancer milieu. Based on the functional relevance of immune networking in tumors, it is advantageous to merge monitoring immune biomarkers into the traditional patient's categorization and treatment regiments, which will provide new prognostic and/or predictive tools to clinical practice. A better identification of environmental and tumor-specific inflammatory mechanisms will allow directing the clinical management of cancer toward a more personalized medicine.
Assuntos
Inflamação/patologia , Células Mieloides/citologia , Neoplasias/patologia , Animais , Antineoplásicos/química , Biomarcadores/metabolismo , Complexo CD3/metabolismo , Granulócitos/citologia , Humanos , Imunossupressores/química , Camundongos , Monócitos/citologia , Prognóstico , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Tumor microenvironment is characterized by chronic inflammation represented by infiltrating leukocytes and soluble mediators, which lead to a local and systemic immunosuppression associated with cancer progression. Here, we used the ret transgenic spontaneous murine melanoma model that mimics human melanoma. Skin tumors and metastatic lymph nodes showed increased levels of inflammatory factors such as IL-1ß, GM-CSF, and IFN-γ, which correlated with tumor progression. Moreover, Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs), known to inhibit tumor reactive T cells, were enriched in melanoma lesions and lymphatic organs during tumor progression. MDSC infiltration was associated with a strong TCR ζ-chain down-regulation in all T cells. Coculturing normal splenocytes with tumor-derived MDSC induced a decreased T-cell proliferation and ζ-chain expression, verifying the MDSC immunosuppressive function and suggesting that the tumor inflammatory microenvironment supports MDSC recruitment and immunosuppressive activity. Indeed, upon manipulation of the melanoma microenvironment with the phosphodiesterase-5 inhibitor sildenafil, we observed reduced levels of numerous inflammatory mediators (e.g., IL-1ß, IL-6, VEGF, S100A9) in association with decreased MDSC amounts and immunosuppressive function, indicating an antiinflammatory effect of sildenafil. This led to a partial restoration of ζ-chain expression in T cells and to a significantly increased survival of tumor-bearing mice. CD8 T-cell depletion resulted in an abrogation of sildenafil beneficial outcome, suggesting the involvement of MDSC and CD8 T cells in the observed therapeutic effects. Our data imply that inhibition of chronic inflammation in the tumor microenvironment should be applied in conjunction with melanoma immunotherapies to increase their efficacy.
Assuntos
Inflamação/imunologia , Melanoma Experimental/imunologia , Animais , Proliferação de Células , Humanos , Tolerância Imunológica/efeitos dos fármacos , Inflamação/genética , Metástase Linfática/imunologia , Sistema Linfático/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-ret/genética , Purinas/farmacologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Citrato de Sildenafila , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Sulfonas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia , Microambiente Tumoral/imunologiaRESUMO
Chronic inflammation is a common factor linking various pathologies that differ in their etiology and physiology such as cancer, autoimmune diseases, and infections. At a certain stage of each of these diseases, while the chronic inflammation proceeds, some key players of the immune system become immunosuppressed as natural killer (NK) cells and T cells. The suppressive environment induced during chronic inflammation is governed by a complex processes characterized by the accumulation and activation of immune suppressor cells, pro-inflammatory cytokines, chemokines, growth and angiogenic factors, and by the activation of several inflammatory signaling pathways mediated predominantly by NFκB and STAT3 transcription factors. A substantial body of evidence supports the notion that the development of a suppressive environment during chronic inflammation limits the success of immune-based and conventional therapies, skewing the balance in favor of a developing pathology. Thus, appropriate, well-designed and fine tuned immune interventions that could resolve inflammatory responses and associated immunosuppression could enhance disease regression and reinforce successful responses to a given therapy. This review describes the interrelationship between chronic inflammation and induced immunosuppression, and explains the current evidence linking inflammation and pathological processes, as found in cancer. We further highlight potential strategies, harnessing the immunosuppressive environment in treating autoimmune diseases and facilitating transplantation. In parallel, we emphasize the use of modalities to combat chronic inflammation-induced immunosuppression in cancer, to enhance the success of immune-based therapies leading to tumor regression. In both cases, the urgent necessity of identifying biomarkers for the evaluation of host immune status is discussed, with the goal of developing optimal personalized treatments.
Assuntos
Tolerância Imunológica , Inflamação/imunologia , Neoplasias/patologia , Animais , Doença Crônica , Humanos , Imunoterapia , Inflamação/metabolismo , Inflamação/terapia , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Terapia de Alvo Molecular , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Evasão Tumoral , Microambiente TumoralRESUMO
Intestinal chronic inflammation is associated with microbial dysbiosis and accumulation of various immune cells including myeloid-derived suppressor cells (MDSC), which profoundly impact the immune microenvironment, perturb homeostasis and increase the risk to develop colitis-associated colorectal cancer (CAC). However, the specific MDSCs-dysbiotic microbiota interactions and their collective impact on CAC development remain poorly understood. In this study, using a murine model of CAC, we demonstrate that CAC-bearing mice exhibit significantly elevated levels of highly immunosuppressive MDSCs, accompanied by microbiota alterations. Both MDSCs and bacteria that infiltrate the colon tissue and developing tumors can be found in close proximity, suggesting intricate MDSC-microbiota cross-talk within the tumor microenvironment. To investigate this phenomenon, we employed antibiotic treatment to disrupt MDSC-microbiota interactions. This intervention yielded a remarkable reduction in intestinal inflammation, decreased MDSC levels, and alleviated immunosuppression, all of which were associated with a significant reduction in tumor burden. Furthermore, we underscore the causative role of dysbiotic microbiota in the predisposition toward tumor development, highlighting their potential as biomarkers for predicting tumor load. We shed light on the intimate MDSCs-microbiota cross-talk, revealing how bacteria enhance MDSC suppressive features and activities, inhibit their differentiation into mature beneficial myeloid cells, and redirect some toward M2 macrophage phenotype. Collectively, this study uncovers the role of MDSC-bacteria cross-talk in impairing immune responses and promoting tumor growth, providing new insights into potential therapeutic strategies for CAC. SIGNIFICANCE: MDSCs-dysbiotic bacteria interactions in the intestine play a crucial role in intensifying immunosuppression within the CAC microenvironment, ultimately facilitating tumor growth, highlighting potential therapeutic targets for improving the treatment outcomes of CAC.
Assuntos
Neoplasias Associadas a Colite , Microbioma Gastrointestinal , Células Supressoras Mieloides , Neoplasias , Animais , Camundongos , Inflamação , Microambiente TumoralRESUMO
Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells known to play a role in perpetuating a wide range of pathologies, such as chronic infections, autoimmune diseases, and cancer. MDSCs were first identified in mice by the markers CD11b+ Gr1+ , and later, based on their morphology, they were classified into two subsets: polymorphonuclear MDSCs, identified by the markers CD11b+ Ly6G+ Ly6CLow , and monocytic MDSCs, detected as being CD11b+ Ly6G- Ly6CHi . MDSCs are studied as immunosuppressive cells in various diseases characterized by chronic inflammation and are associated with disease causes/triggers such as pathogens, autoantigens, and cancer. Therefore, different diseases may diversely affect MDSC metabolism, migration, and differentiation, thus influencing the generated MDSC functional features and ensuing suppressive environment. In order to study MDSCs in a pathology-free environment, we established and calibrated a highly reproducible mouse model that results in the development of chronic inflammation, which is the major cause of MDSC accumulation and immune suppression. The model presented can be used to study MDSC phenotypes, functional diversity, and plasticity. It also permits study of MDSC migration from the bone marrow to peripheral lymphatic and non-lymphatic organs and MDSC crosstalk with extrinsic factors, both in vivo and ex vivo. Furthermore, this model can serve as a platform to assess the effects of anti-MDSC modalities. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Repetitive M.tb immunizations for the induction of chronic inflammation Alternate Protocol 1: Creating a lower grade of inflammation by changing the site of immunization Alternate Protocol 2: In vivo evaluation of immune status Support Protocol 1: Preparation of reconstituted M.tb aliquots and M.tb-IFA emulsions for each of the three injections Support Protocol 2: Preparation of an ovalbumin lentiviral expression vector Support Protocol 3: Fluorescence titering assay for the lentiviral expression vector Support Protocol 4: Spleen excision, tissue dissociation, and preparation of a single-cell suspension Support Protocol 5: Labeling of splenocytes with CFSE proliferation dye.
Assuntos
Células Supressoras Mieloides , Neoplasias , Animais , Autoantígenos/metabolismo , Modelos Animais de Doenças , Inflamação , Camundongos , Células Supressoras Mieloides/metabolismo , Neoplasias/metabolismo , Ovalbumina/metabolismoRESUMO
Myeloid-derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid cells that can be divided into two main subpopulations, polymorphonuclear (PMN) MDSCs and monocytic (M) MDSCs. These cells accumulate during chronic inflammation, characterizing an array of pathologies such as cancer, inflammatory bowel disease, and infectious and autoimmune diseases, and induce immunosuppression. The suppressive effects of MDSCs on the immune system are studied mainly when focusing on their features, functions, and impact on target cells such as T cells, natural killer cells, and B cells, among others. Herein, we describe methods for the analysis of MDSC immunosuppressive features and functions, measuring different mediators that contribute to their activities and how they impact on T cell function. The protocols described are a continuation to those in a companion Current Protocols article by Reuven et al. (2022), which uses a generated single-cell suspension and isolated cells to test their activity. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Evaluating MDSC suppressive features Alternate Protocol 1: Dichlorofluorescein diacetate-based reactive oxygen species detection Support Protocol 1: Detection of nitric oxide secretion Support Protocol 2: Measurement of arginase activity Basic Protocol 2: Evaluating MDSC suppressive function Alternate Protocol 2: In vitro effects of MDSCs on expression of T cell receptor complex during activation Support Protocol 3: Effect of MDSCs on interferon γ production Basic Protocol 3: Effect of MDSCs on T cell proliferation Basic Protocol 4: Effect of MDSCs on T cell cytotoxic activity Alternate Protocol 3: In vivo cytotoxicity assay Basic Protocol 5: Analysis of MDSC differentiation.
Assuntos
Células Supressoras Mieloides , Espécies Reativas de Oxigênio/metabolismo , Arginase/metabolismo , Interferon gama/metabolismo , Óxido Nítrico/metabolismo , Terapia de Imunossupressão , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Myeloid-derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid cells that can be divided into two main subpopulations, polymorphonuclear (PMN) MDSCs and monocytic (M) MDSCs. These cells accumulate during chronic inflammation and induce immunosuppression evident in an array of pathologies such as cancer, inflammatory bowel disease, and infectious and autoimmune diseases. Herein, we describe methods to isolate and characterize MDSCs from various murine tissue, as well as to phenotype blood-derived MDSCs from patients. The protocols describe methods for isolation of total MDSCs and their subpopulations, for characterization, and for evaluation of their distribution within tissue, as well as for assessing their maturation stage by flow cytometry, immunofluorescence analyses, and Giemsa staining. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Single-cell suspension generation from different tissue Alternate Protocol 1: Single-cell suspension generation from subcutaneous melanoma tumors Basic Protocol 2: Characterization of MDSC phenotype Basic Protocol 3: Cell separation using magnetic beads: Separating pan-MDSCs or PMN-MDSC and M-MDSC subpopulations Alternate Protocol 2: Staining and preparing MDSCs for sorting Support Protocol: PMN-MDSC and M-MDSC gating strategy in mouse Basic Protocol 4: Immunofluorescence analysis of MDSCs Basic Protocol 5: Handling human blood samples and characterizing human MDSCs Alternate Protocol 3: Flow cytometry staining of thawed human whole blood samples.
Assuntos
Células Supressoras Mieloides , Animais , Citometria de Fluxo , Humanos , Camundongos , Monócitos , Células Mieloides , FenótipoRESUMO
Elevated osteoclast (OC) activity is a major contributor to inflammatory bone loss (IBL) during chronic inflammatory diseases. However, the specific OC precursors (OCPs) responding to inflammatory cues and the underlying mechanisms leading to IBL are poorly understood. We identified two distinct OCP subsets: Ly6ChiCD11bhi inflammatory OCPs (iOCPs) induced during chronic inflammation, and homeostatic Ly6ChiCD11blo OCPs (hOCPs) which remained unchanged. Functional and proteomic characterization revealed that while iOCPs were rare and displayed low osteoclastogenic potential under normal conditions, they expanded during chronic inflammation and generated OCs with enhanced activity. In contrast, hOCPs were abundant and manifested high osteoclastogenic potential under normal conditions but generated OCs with low activity and were unresponsive to the inflammatory environment. Osteoclasts derived from iOCPs expressed higher levels of resorptive and metabolic proteins than those generated from hOCPs, highlighting that different osteoclast populations are formed by distinct precursors. We further identified the TNF-α and S100A8/A9 proteins as key regulators that control the iOCP response during chronic inflammation. Furthermore, we demonstrated that the response of iOCPs but not that of hOCPs was abrogated in tnf-α-/- mice, in correlation with attenuated IBL. Our findings suggest a central role for iOCPs in IBL induction. iOCPs can serve as potential biomarkers for IBL detection and possibly as new therapeutic targets to combat IBL in a wide range of inflammatory conditions.
RESUMO
Stimulation of TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases promotes tumor progression through numerous cellular mechanisms. TAM cognate ligands GAS6 and PROS1 (for TYRO3 and MERTK) are secreted by host immune cells, an interaction which may support tumor progression. Here, we revealed an unexpected antimetastatic role for myeloid-derived PROS1: suppressing metastatic potential in lung and breast tumor models. Pros1 deletion in myeloid cells led to increased lung metastasis, independent of primary tumor infiltration. PROS1-cKO bone marrow-derived macrophages (BMDMs) led to elevated TNF-α, IL-6, Nos2, and IL-10 via modulation of the Socs3/NF-κB pathway. Conditioned medium from cKO BMDMs enhanced EMT, ERK, AKT, and STAT3 activation within tumor cells and promoted IL-10-dependent invasion and survival. Macrophages isolated from metastatic lungs modulated T cell proliferation and function, as well as expression of costimulatory molecules on DCs in a PROS1-dependent manner. Inhibition of MERTK kinase activity blocked PROS1-mediated suppression of TNF-α and IL-6 but not IL-10. Overall, using lung and breast cancer models, we identified the PROS1/MERTK axis within BMDMs as a potent regulator of adaptive immune responses with a potential to suppress metastatic seeding and revealed IL-10 regulation by PROS1 to deviate from that of TNF-α and IL-6.
Assuntos
Proteínas de Ligação ao Cálcio/imunologia , Interleucina-10/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Mamárias Experimentais/imunologia , Proteínas de Neoplasias/imunologia , Macrófagos Associados a Tumor/imunologia , Animais , Proteínas de Ligação ao Cálcio/genética , Feminino , Interleucina-10/genética , Interleucina-6/genética , Interleucina-6/imunologia , Neoplasias Pulmonares/genética , Neoplasias Mamárias Experimentais/genética , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
SARS-CoV-2 outbreak has been declared by World Health Organization as a worldwide pandemic. However, there are many unknowns about the antigen-specific T-cell-mediated immune responses to SARS-CoV-2 infection. Here, we present both single-cell TCR-seq and RNA-seq to analyze the dynamics of TCR repertoire and immune metabolic functions of blood T cells collected from recently discharged COVID-19 patients. We found that while the diversity of TCR repertoire was increased in discharged patients, it returned to basal level ~1 week after becoming virus-free. The dynamics of T cell repertoire correlated with a profound shift of gene signatures from antiviral response to metabolism adaptation. We also demonstrated that the top expanded T cell clones (~10% of total T cells) display the key anti-viral features in CD8+ T cells, confirming a critical role of antigen-specific T cells in fighting against SARS-CoV-2. Our work provides a basis for further analysis of adaptive immunity in COVID-19 patients, and also has implications in developing a T-cell-based vaccine for SARS-CoV-2.
RESUMO
Intravenous neural precursor cell (NPCs) injection attenuates experimental autoimmune encephalomyelitis by reducing autoreactive T cell encephalitogenicity in lymph nodes in vivo. Here we examined NPC-lymphocyte interactions in vitro. NPCs inhibited the induction of T cell activation marker IL-2-Receptor alpha, ICOS, PD-1 and CTLA-4 and inhibited T cell proliferation. NPCs inhibited T cell activation and proliferation in response to Concavalin-A and to anti-CD3/anti-CD28, which are T cell receptor (TCR)-mediated stimuli, but not in response to phorbol myristate acetate/ionomycin, a TCR-independent stimulus. The suppressive effect was not mediated via downregulation of CD3epsilon or induction of apoptosis. We next examined NPCs effects on inflammatory-cytokine signaling. NPCs impaired IL-2-mediated phosphorylation of JAK3 in lymphocytes, and inhibited IL-6 mediated proliferation of B9 murine hybridoma cells. In conclusion, NPCs ameliorate TCR-mediated T cell activation and inhibit inflammatory cytokines' signaling in immune cells. These findings may underlie the broad anti-inflammatory effects of NPCs in vivo.
Assuntos
Inflamação/metabolismo , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Animais , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Feminino , Interleucina-2/imunologia , Interleucina-6/imunologia , Janus Quinase 3/metabolismo , Linfonodos/citologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Células-Tronco/citologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Myeloid derived suppressor cells (MDSCs) are immature myeloid cells characterized by diverse phenotypes and functions. They impair effector functions of immune cells and promote tumor growth, angiogenesis, and tissue damage. In pathologies characterized by chronic inflammation, MDSCs are arrested in their immature state and migrate from the bone marrow to the periphery and to the site of inflammation, where they mediate immunosuppression. When reaching new environments, which exhibit a different array of cytokines, chemokines, and pro-inflammatory mediators, MDSCs sense and adapt to the altered micro-environment by virtue of acquiring different suppressive features/functions that involve changing their cell fate, surface receptors, metabolism and intracellular as well as secreted molecules. This review summarizes some of the latest publications highlighting various layers of MDSC plasticity in relation to different pathologies. We discuss treatments capitalizing on MDSC plasticity aimed at combating MDSCs or manipulating their suppressive activity for improved therapy.