RESUMO
Group 2 innate lymphoid cells (ILC2s) in the lung are stimulated by inhaled allergens. ILC2s do not directly recognize allergens but they are stimulated by cytokines including interleukin (IL)-33 released by damaged epithelium. In response to allergens, lung ILC2s produce T helper 2 cell type cytokines inducing T cell-independent allergic lung inflammation. Here we examined the fate of lung ILC2s upon allergen challenges. ILC2s proliferated and secreted cytokines upon initial stimulation with allergen or IL-33, and this phase was followed by a contraction phase as cytokine production ceased. Some ILC2s persisted long after the resolution of the inflammation as allergen-experienced ILC2s and responded to unrelated allergens more potently than naive ILC2s, mediating severe allergic inflammation. The allergen-experienced ILC2s exhibited a gene expression profile similar to that of memory T cells. The memory-like properties of allergen-experienced ILC2s may explain why asthma patients are often sensitized to multiple allergens.
Assuntos
Hipersensibilidade/imunologia , Imunidade Inata , Linfócitos/imunologia , Pneumonia/imunologia , Mucosa Respiratória/imunologia , Alérgenos/imunologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Humanos , Memória Imunológica , Mediadores da Inflamação/metabolismo , Interleucina-33/genética , Interleucina-33/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , TranscriptomaRESUMO
Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.
Assuntos
Herpesvirus Humano 1/imunologia , Imunidade Celular , Células Matadoras Naturais/imunologia , Leucemia/imunologia , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação , Degranulação Celular/imunologia , Movimento Celular/imunologia , Feminino , Humanos , Interleucina-15/imunologia , Interleucina-2/imunologia , Células Jurkat , Masculino , NF-kappa B/imunologia , Proteína Quinase C/imunologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologiaRESUMO
Although classical dendritic cells (DCs) arise from distinct progenitors in the bone marrow, the origin of inflammatory DCs and the distinction between monocyte-derived DCs and macrophages is less clear. In vitro culture of mouse bone marrow cells with GM-CSF is a well-established method to generate DCs, but GM-CSF has also been used to generate bone marrow-derived macrophages. In this article, we identify a distinct subpopulation of cells within the GM-CSF bone marrow-derived DC culture based on their ability to bind hyaluronan (HA), a major component of the extracellular matrix and ligand for CD44. HA identified a morphologically distinct subpopulation of cells within the immature DC population (CD11c(+) MHC II(mid/low)) that were CCR5(+)/CCR7(-) and proliferated in response to GM-CSF, but, unlike immature DCs, did not develop into mature DCs expressing CCR7 and high levels of MHC II, even after stimulation with LPS. The majority of these cells produced TNF-α in response to LPS but were unable to activate naive T cells, whereas the majority of mature DCs produced IL-12 and activated naive T cells. This HA binding population shared many characteristics with alveolar macrophages and was retained in the alveolar space after lung instillation even after LPS stimulation, whereas the MHC II(high) mature DCs were found in the draining lymph node. Thus, HA binding in combination with MHC II expression can be used to identify alveolar-like macrophages from GM-CSF-treated bone marrow cultures, which provides a useful in vitro model to study alveolar macrophages.
Assuntos
Células da Medula Óssea/citologia , Linhagem da Célula/imunologia , Células Dendríticas/citologia , Ácido Hialurônico/metabolismo , Macrófagos Alveolares/citologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Ácido Hialurônico/farmacologia , Lipopolissacarídeos/farmacologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Receptores CCR5/genética , Receptores CCR5/imunologia , Receptores CCR7/deficiência , Receptores CCR7/genética , Receptores CCR7/imunologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The human cathelicidin peptide, LL-37, is a host defense peptide with a wide range of immunomodulatory activities and modest direct antimicrobial properties. LL-37 can exert both pro- and anti-inflammatory effects and can modulate the proinflammatory responses of human peripheral blood monocytes and epithelial cells. In this study, we evaluated the effect of LL-37 on mouse bone marrow-derived macrophages (BMDM) and tissue macrophages in vitro and in vivo. LL-37 dramatically reduced TNF-α and NO levels produced by LPS and IFN-γ-polarized M1-BMDM and slightly reduced reactive oxygen species production by these cells. LL-37 did not affect the ability of IL-4-polarized M2-BMDM to upregulate arginase activity, although it did inhibit LPS-induced TNF-α secretion in these cells. LL-37 did not compromise the ability of M1-polarized BMDM to phagocytose and kill bacteria and did not affect the uptake of apoptotic neutrophils by M2-polarized BMDM. However, LL-37-treated M1-BMDM were more efficient at suppressing tumor growth in vitro. LL-37 significantly reduced LPS-induced TNF-α secretion in ex vivo alveolar macrophages, whereas its effect on peritoneal macrophages was much less dramatic. Effective inhibition of LPS-induced TNF-α secretion by alveolar macrophages also occurred in vivo when LL-37 was administered by intratracheal injection. This demonstrates a selective ability of LL-37 to decrease M1-BMDM, M2-BMDM, and tissue macrophage production of the proinflammatory cytokine TNF-α in response to LPS while leaving other crucial anti-inflammatory M1 and M2 macrophage functions unaltered.
Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Diferenciação Celular/imunologia , Separação Celular , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Inflamação/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Fagocitose/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia , CatelicidinasRESUMO
CD44 is a cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is involved in processes ranging from leukocyte recruitment to wound healing. In the immune system, the binding of hyaluronan to CD44 is tightly regulated, and exposure of human peripheral blood monocytes to inflammatory stimuli increases CD44 expression and induces hyaluronan binding. Here we sought to understand how mouse macrophages regulate hyaluronan binding upon inflammatory and anti-inflammatory stimuli. Mouse bone marrow-derived macrophages stimulated with tumor necrosis factor α or lipopolysaccharide and interferon-γ (LPS/IFNγ) induced hyaluronan binding by up-regulating CD44 and down-regulating chondroitin sulfation on CD44. Hyaluronan binding was induced to a lesser extent in interleukin-4 (IL-4)-activated macrophages despite increased CD44 expression, and this was attributable to increased chondroitin sulfation on CD44, as treatment with ß-d-xyloside to prevent chondroitin sulfate addition significantly enhanced hyaluronan binding. These changes in the chondroitin sulfation of CD44 were associated with changes in mRNA expression of two chondroitin sulfotransferases, CHST3 and CHST7, which were decreased in LPS/IFNγ-stimulated macrophages and increased in IL-4-stimulated macrophages. Thus, inflammatory and anti-inflammatory stimuli differentially regulate the chondroitin sulfation of CD44, which is a dynamic physiological regulator of hyaluronan binding by CD44 in mouse macrophages.
Assuntos
Sulfatos de Condroitina/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Interleucina-4/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Animais , Linhagem Celular Tumoral , Sulfatos de Condroitina/imunologia , Regulação da Expressão Gênica , Humanos , Receptores de Hialuronatos/imunologia , Ácido Hialurônico/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interferon gama/farmacologia , Interleucina-4/imunologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Sulfotransferases/imunologia , Sulfotransferases/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Carboidrato SulfotransferasesRESUMO
CD44 is expressed on T cells where its ability to bind hyaluronan is tightly regulated. Here, we investigated when T cells bind hyaluronan during an immune response. We found that naïve, murine T cells do not bind fluoresceinated hyaluronan but are induced to bind upon antigen-induced T-cell activation in vitro and in vivo. Hyaluronan binding occurred on proliferating T cells and the percentage of hyaluronan-binding cells correlated with the strength of the activation stimulus. A small percentage of hyaluronan-binding cells persisted after in vitro activation and had a memory phenotype (CD122(+) CD44(hi)). This hyaluronan-binding population increased after culture with IL-7 or IL-15 and proliferated more rapidly than nonbinding cells. In vivo, approximately 20-30% of antigen-specific OT-I CD8(+) memory T cells in the spleen and BM bound hyaluronan. Hyaluronan binding identified memory cells that proliferated faster in IL-7 and IL-15, and enriched for CD62L(+) central memory cells. In vivo homeostatic proliferation induced hyaluronan binding on a small percentage of the most rapidly dividing cells after several cell divisions. This study demonstrates that hyaluronan binding is induced upon antigen-induced T-cell activation and occurs on a percentage of the most proliferative activated and memory T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Ácido Hialurônico/imunologia , Memória Imunológica , Ativação Linfocitária , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Homeostase , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Alveolar macrophages (AMs) are CD44 expressing cells that reside in the alveolar space where they maintain lung homeostasis by serving critical roles in immunosurveillance and lipid surfactant catabolism. AMs lacking CD44 are unable to bind the glycosaminoglycan, hyaluronan, which compromises their survival and leads to reduced numbers of AMs in the lung. Using RNA sequencing, lipidomics and multiparameter flow cytometry, we demonstrate that CD44-/- mice have impaired AM lipid homeostasis and increased surfactant lipids in the lung. CD44-/- AMs had increased expression of CD36, a lipid scavenger receptor, as well as increased intracellular lipid droplets, giving them a foamy appearance. RNA sequencing revealed the differential expression of genes associated with lipid efflux and metabolism in CD44-/- AMs. Lipidomic analysis showed increased lipids in both the supernatant and cell pellet extracted from the bronchoalveolar lavage of CD44-/- mice. Phosphatidylcholine species, cholesterol, oxidized phospholipids and levels of reactive oxygen species (ROS) were increased in CD44-/- AMs. Oxidized phospholipids were more cytotoxic to CD44-/- AMs and induced greater lung inflammation in CD44-/- mice. Reconstitution of CD44+/+ mice with CD44-/- bone marrow as well as adoptive transfer of CD44-/- AMs into CD44+/+ mice showed that lipid accumulation in CD44-/- AMs occurred irrespective of the lung environment, suggesting a cell intrinsic defect. Administration of colony stimulating factor 2 (CSF-2), a critical factor in AM development and maintenance, increased AM numbers in CD44-/- mice and decreased phosphatidylcholine levels in the bronchoalveolar lavage, but was unable to decrease intracellular lipid accumulation in CD44-/- AMs. Peroxisome proliferator-activated receptor gamma (PPARγ), downstream of CSF-2 signaling and a regulator of lipid metabolism, was reduced in the nucleus of CD44-/- AMs, and PPARγ inhibition in normal AMs increased their lipid droplets. Thus, CD44 deficiency causes defects in AMs that lead to abnormal lipid accumulation and oxidation, which exacerbates oxidized lipid-induced lung inflammation. Collectively, these findings implicate CD44 as a regulator of lung homeostasis and inflammation.
Assuntos
Colesterol/metabolismo , Homeostase/genética , Receptores de Hialuronatos/metabolismo , Macrófagos Alveolares/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Pneumonia/metabolismo , Animais , Antígenos CD36/metabolismo , Feminino , Receptores de Hialuronatos/genética , Gotículas Lipídicas/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , PPAR gama/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genéticaRESUMO
Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.