RESUMO
BACKGROUND: Solid tumors produce proteins that can induce the accumulation of bone marrow-derived cells in various tissues, and these cells can enhance metastatic tumor growth by several mechanisms. 4T1 murine mammary tumors are known to produce granulocyte colony-stimulating factor (G-CSF) and increase the numbers of immunosuppressive CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) in tissues such as the spleen and lungs of tumor-bearing mice. While surgical resection of primary tumors decreases MDSC levels in the spleen, the longevity and impact of MDSCs and other immune cells in the lungs after tumor resection have been less studied. METHODS: We used mass cytometry time of flight (CyTOF) and flow cytometry to quantify MDSCs in the spleen, peripheral blood, and lungs of mice bearing orthotopic murine mammary tumors. We also tested the effect of primary tumor resection and/or gemcitabine treatment on the levels of MDSCs, other immune suppressor and effector cells, and metastatic tumor cells in the lungs. RESULTS: We have found that, similar to mice with 4T1 tumors, mice bearing metastatic 4T07 tumors also exhibit accumulation of CD11b+Gr1+ MDSCs in the spleen and lungs, while tissues of mice with non-metastatic 67NR tumors do not contain MDSCs. Mice with orthotopically implanted 4T1 tumors have increased granulocytic (G-) MDSCs, monocytic (M-) MDSCs, macrophages, eosinophils, and NK cells in the lungs. Resection of primary 4T1 tumors decreases G-MDSCs, M-MDSCs, and macrophages in the lungs within 48 h, but significant numbers of functional immunosuppressive G-MDSCs persist in the lungs for 2 weeks after tumor resection, indicative of an environment that can promote metastatic tumor growth. The chemotherapeutic agent gemcitabine depletes G-MDSCs, M-MDSCs, macrophages, and eosinophils in the lungs of 4T1 tumor-bearing mice, and we found that treating mice with gemcitabine after primary tumor resection decreases residual G-MDSCs in the lungs and decreases subsequent metastatic growth. CONCLUSIONS: Our data support the development of therapeutic strategies to target MDSCs and to monitor MDSC levels before and after primary tumor resection to enhance the effectiveness of immune-based therapies and improve the treatment of metastatic breast cancer in the clinic.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Neoplasias Mamárias Experimentais/patologia , Mastectomia , Células Supressoras Mieloides/efeitos dos fármacos , Animais , Antígenos Ly/metabolismo , Antígeno CD11b/metabolismo , Linhagem Celular Tumoral , Terapia Combinada , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Eosinófilos/patologia , Feminino , Células Matadoras Naturais/patologia , Neoplasias Pulmonares/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Células Supressoras Mieloides/imunologia , GencitabinaRESUMO
Lung cancer is the leading cause of cancer mortality worldwide, and at only 18%, it has one of the lowest 5-year survival rates of all malignancies. With its highly complex mutational landscape, treatment strategies against lung cancer have proved largely ineffective. However with the recent success of immunotherapy trials in lung cancer, there is renewed enthusiasm in targeting the immune component of tumors. Macrophages make up the majority of the immune infiltrate in tumors and are a key cell type linking inflammation and cancer. Although the mechanisms through which inflammation promotes cancer are not fully understood, two connected hypotheses have emerged: an intrinsic pathway, driven by genetic alterations that lead to neoplasia and inflammation, and an extrinsic pathway, driven by inflammatory conditions that increase cancer risk. Here, we discuss the contribution of macrophages to these pathways and subsequently their roles in established tumors. We highlight studies investigating the association of macrophages with lung cancer prognosis and discuss emerging therapeutic strategies for targeting macrophages in the tumor microenvironment.
Assuntos
Imunoterapia/métodos , Inflamação/etiologia , Neoplasias Pulmonares/terapia , Macrófagos/imunologia , Biomarcadores Tumorais/análise , Ensaios Clínicos como Assunto , Progressão da Doença , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Fenótipo , Prognóstico , Taxa de SobrevidaRESUMO
SH2-containing-inositol-5'-phosphatase (SHIP) is a negative regulator of the phosphatidylinositol-3-kinase pathway in hematopoietic cells and limits the development of leukemias and lymphomas. The potential role of SHIP in solid tumor development and metastasis remains unknown. While SHIP restricts the aberrant development of myeloid cells in C57BL/6 mice, there are conflicting reports regarding the effect of SHIP deletion in BALB/c mice with important consequences for determining the influence of SHIP in different model tumor systems. We generated SHIP-/- BALB/c mice and challenged them with syngeneic non-metastatic 67NR or metastatic 4T1 mammary tumors. We demonstrate that SHIP restricts the development, alternative-activation, and immunosuppressive function of myeloid cells in tumor-free and tumor-bearing BALB/c mice. Tumor-free SHIP-/- BALB/c mice exhibited pulmonary inflammation, myeloid hyperplasia, and M2-polarized macrophages and this phenotype was greatly exacerbated by 4T1, but not 67NR, tumors. 4T1-bearing SHIP-/- mice rapidly lost weight and died from necrohemorrhagic inflammatory pulmonary disease, characterized by massive infiltration of pulmonary macrophages and myeloid-derived suppressor cells that were more M2-polarized and immunosuppressive than wild-type cells. Importantly, while SHIP loss did not affect primary tumor growth, 4T1-bearing SHIP-/- mice had 7.5-fold more metastatic tumor cells in their lungs than wild-type mice, consistent with the influence of immunosuppressive myeloid cells on metastatic growth. Our findings identify the hematopoietic cell-restricted protein SHIP as an intriguing target to influence the development of solid tumor metastases, and support development of SHIP agonists to prevent the accumulation of immunosuppressive myeloid cells and tumor metastases in the lungs to improve treatment of metastatic breast cancer.
Assuntos
Neoplasias Pulmonares/prevenção & controle , Neoplasias Mamárias Experimentais/prevenção & controle , Monoéster Fosfórico Hidrolases/fisiologia , Pneumonia/prevenção & controle , Animais , Apoptose , Western Blotting , Proliferação de Células , Feminino , Humanos , Técnicas Imunoenzimáticas , Inositol Polifosfato 5-Fosfatases , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Macrófagos/metabolismo , Macrófagos/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Células Mieloides/patologia , Pneumonia/genética , Pneumonia/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mobilization of bone marrow-derived cells (BMDC) to distant tissues before the arrival of disseminated tumor cells has been shown preclinically to facilitate metastasis through the establishment of metastatic niches. Primary tumor hypoxia has been demonstrated to play a pivotal role in the production of chemokines and cytokines responsible for the mobilization of these BMDCs, especially in breast cancer. Carbonic anhydrase IX (CAIX, CA9) expression is highly upregulated in hypoxic breast cancer cells through the action of hypoxia-inducible factor-1 (HIF1). Preclinical evidence has demonstrated that CAIX is required for breast tumor growth and metastasis; however, the mechanism by which CAIX exerts its prometastatic function is not well understood. Here, we show that CAIX is indispensable for the production of granulocyte colony-stimulating factor (G-CSF) by hypoxic breast cancer cells and tumors in an orthotopic model. Furthermore, we demonstrate that tumor-expressed CAIX is required for the G-CSF-driven mobilization of granulocytic myeloid-derived suppressor cells (MDSC) to the breast cancer lung metastatic niche. We also determined that CAIX expression is required for the activation of NF-κB in hypoxic breast cancer cells and constitutive activation of the NF-κB pathway in CAIX-depleted cells restored G-CSF secretion. Together, these findings identify a novel hypoxia-induced CAIX-NF-κB-G-CSF cellular signaling axis culminating in the mobilization of granulocytic MDSCs to the breast cancer lung metastatic niche.
Assuntos
Anidrases Carbônicas/fisiologia , Movimento Celular , Fator Estimulador de Colônias de Granulócitos/biossíntese , Células Mieloides/fisiologia , Neoplasias Experimentais/patologia , Animais , Antígeno CD11b/análise , Anidrase Carbônica IX , Hipóxia Celular , Quimiocina CXCL10/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/fisiologia , Metástase NeoplásicaRESUMO
We recently demonstrated that both murine and human carcinomas grow significantly slower in mice on low carbohydrate (CHO), high protein diets than on isocaloric Western diets and that a further reduction in tumor growth rates occur when the low CHO diets are combined with the cyclooxygenase-2 inhibitor, celecoxib. Following upon these studies, we asked herein what effect low CHO, high protein diets, with or without celecoxib, might have on tumor metastasis. In the highly metastatic 4T1 mouse mammary tumor model, a 15% CHO, high protein diet supplemented with celecoxib (1 g/kg chow) markedly reduced lung metastases. Moreover, in longer-term studies using male Transgenic Adenocarcinoma of the Mouse Prostate mice, which are predisposed to metastatic prostate cancer, the 15% CHO diet, with and without celecoxib (0.3 g/kg chow), gave the lowest incidence of metastases, but a more moderate 25% CHO diet containing celecoxib led to the best survival. Metabolic studies with 4T1 tumors suggested that the low CHO, high protein diets may be forcing tumors to become dependent on amino acid catabolism for survival/growth. Taken together, our results suggest that a combination of a low CHO, high protein diet with celecoxib substantially reduces metastasis.
Assuntos
Dieta com Restrição de Carboidratos , Proteínas Alimentares/farmacologia , Metástase Neoplásica/tratamento farmacológico , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Animais , Celecoxib , Dietoterapia/métodos , Modelos Animais de Doenças , Neoplasias Pulmonares/dietoterapia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Metástase Neoplásica/terapia , Neoplasias da Próstata/dietoterapia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Myeloid-derived suppressor cells (MDSCs) are emerging as potential promoters of metastatic tumor growth, and there is interest in targeting immature MDSCs by inducing their differentiation into more mature myeloid cells. We used all-trans retinoic acid (ATRA) to differentiate MDSCs in mice bearing metastatic 4T1 or 4TO7 murine mammary tumors, and assessed the immune-suppressive mechanisms and potencies of different myeloid cell subpopulations. Metastatic mammary tumors induced the accumulation of distinct populations of immature CD11b(+)Gr1(+)F4/80(-)Ly6C(mid)Ly6G(+) MDSCs ("Gr1(+) cells") and mature CD11b(+)Gr1(-)F4/80(+) cells ("F4/80(+) cells") in metastatic target organs. ATRA triggered the differentiation of Gr1(+) cells into F4/80(+) cells in the lungs and, unexpectedly, enhanced pulmonary metastatic tumor growth. We found that F4/80(+)Ly6C(-)Ly6G(-) mature macrophages (Ms) were up to 30-fold more potent immune suppressors than Gr1(+) cells on a per-cell basis, which we postulate may contribute to the increased metastatic growth observed with ATRA treatment. F4/80(+) cells and Gr1(+) cells used different reactive oxygen species (ROS)-mediated mechanisms of immunosuppression ex vivo, with F4/80(+) cells producing higher levels of ROS, which is consistent with their superior immunosuppressive abilities. These data highlight the potent immunosuppressive functions of Ms, reveal that Ms can suppress T cell responses via ROS production, and suggest that ROS inhibitors may be useful in promoting antitumor immune responses. Our findings also caution against using ATRA to modulate myeloid cell differentiation and function to treat breast cancer metastases in the lung, and support the development of therapeutic strategies to enhance antitumor immunity by targeting myeloid cells as a collective group.
Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Macrófagos/imunologia , Células Mieloides/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Imunofenotipagem , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Células Mieloides/patologia , Metástase Neoplásica , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tretinoína/farmacologiaRESUMO
As more groups investigate the role of myeloid-derived suppressor cells (MDSCs) in promoting the growth of primary tumors and distant tumor metastases, it is imperative to ensure the accurate detection and quantification of MDSC immunosuppression ex vivo. MDSCs are defined by their ability to suppress immune responses. Although different in vitro culture conditions have been used to study MDSCs, the effect of different culture conditions on MDSC immunosuppression is unknown. We therefore isolated MDSCs from the lungs and spleens of 4T1 murine mammary tumor-bearing mice and assayed MDSC-mediated suppression of T cell responses under different culture conditions. We found that 4T1-induced MDSCs effectively suppressed T cell proliferation under serum-free conditions, but not when fetal calf serum (FCS) was present. FCS neither altered the immunosuppressive activities of other myeloid cell types (i.e., peritoneal or tumor-associated macrophages) nor modified the susceptibility of T cells to myeloid cell-mediated suppression, but instead acted directly on 4T1-induced MDSCs to significantly reduce their immunosuppressive function. Importantly, we found that bovine serum albumin was a major contributor to the antagonistic effects of FCS on 4T1-induced MDSC immunosuppression by inhibiting reactive oxygen species production from MDSCs. This work reveals that in vitro culture conditions influence the immunosuppressive properties of MDSCs and highlights the importance of testing different culture conditions on MDSC phenotype to ensure that MDSC immunosuppression is not being masked. These data have important implications for the accurate detection and identification of MDSCs, as well as for determining the influence of MDSC-mediated immunosuppression on primary and metastatic tumor growth.
Assuntos
Neoplasias Mamárias Experimentais/sangue , Neoplasias Mamárias Experimentais/imunologia , Células Mieloides/imunologia , Animais , Bovinos , Técnicas de Cultura de Células , Processos de Crescimento Celular/imunologia , Feminino , Terapia de Imunossupressão , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Soroalbumina Bovina/imunologia , Soroalbumina Bovina/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
4T1 murine mammary carcinoma cells implanted in syngeneic Balb/c mice are increasingly being used in metastasis research, with some groups using this model to study tumor-induced accumulation of bone marrow-derived cells in metastatic target organs. Bone marrow-derived cells (including CD11b(+)Gr-1(+) myelomonocytic cells) are thought to modify the local lung microenvironment to facilitate subsequent colonization by metastatic tumor cells. While quantification of metastatic 4T1 tumor cells in various tissues can be done using ex vivo colony-forming assays, detection of metastatic 4T1 cells is often facilitated by expressing fluorescent proteins in the tumor cells prior to implantation. We found that Balb/c mice mount a potent immune response against 4T1 cells expressing green fluorescent protein (GFP) that includes the generation of anti-GFP antibodies in the circulation. Importantly, the number of bone marrow-derived CD11b(+)Gr-1(+) cells and metastatic tumor cells that accumulate in the lungs is significantly decreased in mice implanted with 4T1 cells expressing GFP compared with mice bearing wild-type 4T1 tumors. Taken together, our data caution against the use of GFP-expressing tumor cells in the Balb/c mouse strain, particularly for studying the influence of immunomodulatory cells on tumor cell metastasis.
Assuntos
Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Pulmão/imunologia , Neoplasias Mamárias Experimentais/imunologia , Células Mieloides/imunologia , Animais , Antígeno CD11b , Feminino , Proteínas de Fluorescência Verde/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Dendritic cells (DCs) not only play a crucial role in activating immune cells but also suppressing them. We recently investigated SHIP's role in murine DCs in terms of immune cell activation and found that TLR agonist-stimulated SHIP-/- GM-CSF-derived DCs (GM-DCs) were far less capable than wild type (WT, SHIP+/+) GM-DCs at activating T cell proliferation. This was most likely because SHIP-/- GM-DCs could not up-regulate MHCII and/or co-stimulatory receptors following TLR stimulation. However, the role of SHIP in DC-induced T cell suppression was not investigated. METHODOLOGY/PRINCIPAL FINDINGS: In this study we examined SHIP's role in DC-induced T cell suppression by co-culturing WT and SHIP-/- murine DCs, derived under different conditions or isolated from spleens, with αCD3+ αCD28 activated WT T cells and determined the relative suppressive abilities of the different DC subsets. We found that, in contrast to SHIP+/+ and -/- splenic or Flt3L-derived DCs, which do not suppress T cell proliferation in vitro, both SHIP+/+ and -/- GM-DCs were capable of potently suppressing T cell proliferation. However, WT GM-DC suppression appeared to be mediated, at least in part, by nitric oxide (NO) production while SHIP-/- GM-DCs expressed high levels of arginase 1 and did not produce NO. Following exhaustive studies to ascertain the mechanism of SHIP-/- DC-mediated suppression, we could conclude that cell-cell contact was required and the mechanism may be related to their relative immaturity, compared to SHIP+/+ GM-DCs. CONCLUSIONS: These findings suggest that although both SHIP+/+ and -/- GM-DCs suppress T cell proliferation, the mechanism(s) employed are different. WT GM-DCs suppress, at least in part, via IFNγ-induced NO production while SHIP-/- GM-DCs do not produce NO and suppression can only be alleviated when contact is prevented.
Assuntos
Células Dendríticas/citologia , Monoéster Fosfórico Hidrolases/deficiência , Linfócitos T/citologia , Aminoácidos/metabolismo , Animais , Arginase/metabolismo , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/enzimologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Terapia de Imunossupressão , Inositol Polifosfato 5-Fosfatases , Interferon gama/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Modelos Imunológicos , Óxido Nítrico/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
The SH2-containing inositol-5'-phosphatase, SHIP (or SHIP1), is a hematopoietic-restricted phosphatidylinositide phosphatase that translocates to the plasma membrane after extracellular stimulation and hydrolyzes the phosphatidylinositol-3-kinase-generated second messenger PI-3,4,5-P(3) to PI-3,4-P(2). As a result, SHIP dampens down PI-3,4,5-P(3)-mediated signaling and represses the proliferation, differentiation, survival, activation, and migration of hematopoietic cells. There are multiple lines of evidence suggesting that SHIP may act as a tumor suppressor during leukemogenesis and lymphomagenesis. Because of its ability to skew macrophage progenitors toward M1 macrophages and naïve T cells toward T helper 1 and T helper 17 cells, SHIP may play a critical role in activating the immune system to eradicate solid tumors. In this review, we will discuss the role of SHIP in hematopoietic cells and its therapeutic potential in terms of suppressing leukemias and lymphomas and manipulating the immune system to combat cancer.
Assuntos
Neoplasias/fisiopatologia , Monoéster Fosfórico Hidrolases/fisiologia , Animais , Humanos , Inositol Polifosfato 5-Fosfatases , Camundongos , Camundongos Knockout , Neoplasias/enzimologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Conformação ProteicaRESUMO
Resident tissue macrophages (Mφs) continually survey the microenvironment, ingesting Ags and presenting them on their surface for recognition by T cells. Because these Ags can be either host cell- or pathogen-derived, Mφs must be able to distinguish whether a particular Ag should provoke an immune response or be tolerated. However, the mechanisms that determine whether Mφs promote or inhibit T cell activation are not well understood. To investigate this, we first determined the mechanism by which murine resident peritoneal Mφs suppress in vitro T cell proliferation in the absence of pathogens and then explored the effects of different pathogen-derived molecules on Mφ immunosuppression. Our results suggest that, in response to IFN-γ, which is secreted by TCR-activated T cells, resident peritoneal Mφs acquire immunosuppressive properties that are mediated by NO. However, pretreatment of Mφs with LPS or dsRNA, but not CpG or peptidoglycan, eliminates their suppressive properties, in part via the induction of autocrine-acting IFN-ß. These results suggest TLR agonists that activate TRIF, and consequently induce IFN-ß, but not those that exclusively signal through MyD88, abrogate the immunosuppressive properties of Mφs, and thus promote T cell expansion and elimination of invading microorganisms.
Assuntos
Tolerância Imunológica/imunologia , Interferon beta/biossíntese , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Western Blotting , Proliferação de Células , Separação Celular , Citometria de Fluxo , Interferon beta/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA de Cadeia Dupla/imunologia , Linfócitos T/metabolismo , Receptores Toll-Like/agonistasRESUMO
There is a great deal of interest in determining what regulates the generation of classically activated (M1) vs alternatively activated (M2) macrophages (Mphis) because of the opposing effects that these two Mphi subsets have on tumor progression. We show herein that IL-3 and, to a lesser extent, GM-CSF skew murine Mphi progenitors toward an M2 phenotype, especially in the absence of SHIP. Specifically, the addition of these cytokines, with or without M-CSF, to adherence- or lineage-depleted (Lin(-)) SHIP(-/-) bone marrow (BM) cells induces high levels of the M2 markers, arginase I, and Ym1 in the resulting mature Mphis. These in vitro-derived mature Mphis also display other M2 characteristics, including an inability to enhance anti-CD3-stimulated splenic T cell secretion of IFN-gamma and low IL-12 and high IL-10 production in response to LPS. Not surprisingly, given that IL-3 and GM-CSF utilize STAT5 to trigger many downstream signaling pathways, this M2 phenotype is suppressed when STAT5(-/-) BM cells are used. Unexpectedly, however, this M2 phenotype is also suppressed when STAT6(-/-) BM cells are used, suggesting that IL-4- or IL-13-induced signaling might be involved. Consistent with this, we found that IL-3 and GM-CSF stimulate the production of IL-4, especially from SHIP(-/-) Lin(-) BM cells, and that neutralizing anti-IL-4 Abs block IL-3-induced M2 skewing. Moreover, we found that basophil progenitors within the Lin(-) BM are responsible for this IL-3- and GM-CSF-induced IL-4 production, and that SHIP represses M2 skewing not by preventing skewing within Mphis themselves but by inhibiting IL-4 production from basophils.
Assuntos
Basófilos/metabolismo , Diferenciação Celular/imunologia , Interleucina-3/farmacologia , Interleucina-4/biossíntese , Macrófagos/citologia , Monoéster Fosfórico Hidrolases/fisiologia , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Inositol Polifosfato 5-Fosfatases , Ativação de Macrófagos , Camundongos , Células-Tronco/citologiaRESUMO
Maintaining an appropriate balance between subsets of CD4(+) Th and T regulatory cells (Tregs) is critical to maintain immune homeostasis and prevent autoimmunity. Through a common requirement for TGF-beta, the development of peripherally induced Tregs is intimately linked to that of Th17 cells, with the resulting lineages depending on the presence of proinflammatory cytokines such as IL-6. Currently very little is known about the molecular signaling pathways that control the development of Tregs vs Th17 cells. Reduced activity of the PI3K pathway is required for TGF-beta-mediated induction of Foxp3 expression and the suppressive activity of Tregs. To investigate how negative regulators of the PI3K pathway impact Treg development, we investigated whether SHIP, a lipid phosphatase that regulates PI3K activity, also plays a role in the development and function of Tregs. SHIP-deficient Tregs maintained suppressive capacity in vitro and in a T cell transfer model of colitis. Surprisingly, SHIP-deficient Th cells were significantly less able to cause colitis than were wild-type Th cells due to a profound deficiency in Th17 cell differentiation, both in vitro and in vivo. The inability of SHIP-deficient T cells to develop into Th17 cells was accompanied by decreased IL-6-stimulated phosphorylation of STAT3 and an increased capacity to differentiate into Treg cells under the influence of TGF-beta and retinoic acid. These data indicate that SHIP is essential for normal Th17 cell development and that this lipid phosphatase plays a key role in the reciprocal regulation of Tregs and Th17 cells.
Assuntos
Homeostase/imunologia , Interleucina-17 , Monoéster Fosfórico Hidrolases/fisiologia , Subpopulações de Linfócitos T/citologia , Linfócitos T Reguladores/citologia , Animais , Diferenciação Celular , Colite/etiologia , Inositol Polifosfato 5-Fosfatases , Interleucina-6/farmacologia , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/deficiência , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/transplante , Linfócitos T Reguladores/transplanteRESUMO
Gram-negative bacterial infections, unlike viral infections, do not typically protect against subsequent viral infections. This is puzzling given that lipopolysaccharide (LPS) and double-stranded (ds) RNA both activate the TIR domain-containing adaptor-inducing interferon beta (TRIF) pathway and, thus, are both capable of eliciting an antiviral response by stimulating type I interferon (IFN) production. We demonstrate herein that SH2-containing inositol-5'-phosphatase (SHIP) protein levels are dramatically increased in murine macrophages via the MyD88-dependent pathway, by up-regulating autocrine-acting transforming growth factor-beta (TGFbeta). The increased SHIP then mediates, via inhibition of the phosphatidylinositol-3-kinase (PI3K) pathway, cytosine-phosphate-guanosine (CPG)- and LPS-induced tolerance and cross-tolerance and restrains IFN-beta production induced by a subsequent exposure to LPS or dsRNA. Intriguingly, we found, using isoform-specific PI3K inhibitors, that LPS- or cytosine-phosphate-guanosine-induced interleukin-6 (IL-6) is positively regulated by p110alpha, -gamma, and -delta but negatively regulated by p110beta. This may explain some of the controversy concerning the role of PI3K in Toll-like receptor-induced cytokine production. Consistent with our in vitro findings, SHIP(-/-) mice overproduce IFN-beta in response to LPS, and this leads to antiviral hypothermia. Thus, up-regulation of SHIP in response to Gram-negative bacterial infections probably explains the inability of such infections to protect against subsequent viral infections.
Assuntos
Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Lipopolissacarídeos/farmacologia , Monoéster Fosfórico Hidrolases/genética , Vírus/imunologia , Animais , Células Cultivadas , Ilhas de CpG/imunologia , Ilhas de CpG/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Hipotermia/genética , Hipotermia/imunologia , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/genética , Inositol Polifosfato 5-Fosfatases , Interferon beta/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
BACKGROUND AND OBJECTIVES: Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo. DESIGN AND METHODS: ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers. Subsets of these cells were then isolated by multi-parameter flow cytometry and assessed for short-and long-term repopulating activity in sublethally irradiated immunodeficient mice. RESULTS: Most short-term myeloid repopulating cells (STRC-M) and all long-term lympho-myeloid repopulating cells (LTRC-ML) stained selectively as ALDH+. Limiting dilution analysis of the frequencies of both STRC-M and LTRC-ML showed that they were similarly and most highly enriched in the 10% top ALDH+ cells. Removal of cells expressing CD2, CD3, CD7, CD14, CD16, CD24, CD36, CD38, CD56, CD66b, or glycophorin A from the ALDH+ low-density fraction of human cord blood cells with low light side-scattering properties yielded a population containing LTRC-ML at a frequency of 1/360. INTERPRETATION AND CONCLUSION: Elevated ALDH activity is a broadly inclusive property of primitive human cord blood cells that, in combination with other markers, allows easy isolation of the stem cell fraction at unprecedented purities.
Assuntos
Aldeído Desidrogenase/metabolismo , Separação Celular/métodos , Células-Tronco Hematopoéticas/enzimologia , Animais , Células da Medula Óssea/enzimologia , Sangue Fetal/enzimologia , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
The SH2-containing inositol-5'-phosphatase, SHIP, represses the proliferation, survival, and activation of hematopoietic cells, in large part by translocating to membranes following extracellular stimulation and hydrolysing the phosphatidylinositol-3-kinase (PI3K)-generated second messenger PI-3,4,5-P3 (PIP3) to PI-3,4-P2. SHIP-/- mice have, as a result, an increased number of monocyte/macrophages because their progenitors display enhanced survival and proliferation, as well as more rapid differentiation. Interestingly, SHIP-/- mice do not display lipopolysaccharide (LPS)- or CpG oligonucleotide-induced tolerance because this blunting of inflammatory mediator production is contingent upon LPS- and CpG-induced upregulation of SHIP in their macrophages and mast cells. This upregulation is mediated via the production of autocrine-acting TGFbeta which is induced via the MyD88-dependent pathway. The increased levels of SHIP then inhibit both MyD88-dependent and independent signaling. Intriguingly, SHIP-/- peritoneal and alveolar macrophages produce less nitric oxide (NO) than wild-type macrophages because they have constitutively high arginase I levels and this enzyme competes with inducible nitric oxide synthase (iNOS) for the substrate L-arginine. It is likely that, in the face of chronically elevated PIP3 levels in their myeloid progenitors, SHIP-/- mice display a skewed development away from M1 (killer) macrophages towards M2 (healing) macrophages. This suggests that SHIP plays a critical role in programming macrophages.
Assuntos
Macrófagos/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Animais , Tolerância Imunológica/fisiologia , Inositol Polifosfato 5-Fosfatases , Camundongos , Camundongos Knockout , Fenótipo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genéticaRESUMO
Heterogeneity in the differentiation behavior of hematopoietic stem cells is well documented but poorly understood. To investigate this question at a clonal level, we isolated a subpopulation of adult mouse bone marrow that is highly enriched for multilineage in vivo repopulating cells and transplanted these as single cells, or their short-term clonal progeny generated in vitro, into 352 recipients. Of the mice, 93 showed a donor-derived contribution to the circulating white blood cells for at least 4 months in one of four distinct patterns. Serial transplantation experiments indicated that two of the patterns were associated with extensive self-renewal of the original cell transplanted. However, within 4 days in vitro, the repopulation patterns subsequently obtained in vivo shifted in a clone-specific fashion to those with less myeloid contribution. Thus, primitive hematopoietic cells can maintain distinct repopulation properties upon serial transplantation in vivo, although these properties can also alter rapidly in vitro.