RESUMEN
Granzymes are a family of serine proteases mainly expressed by CD8+ T cells, natural killer cells, and innate-like lymphocytes1,2. Although their major role is thought to be the induction of cell death in virally infected and tumor cells, accumulating evidence suggests some granzymes can regulate inflammation by acting on extracellular substrates2. Recently, we found that the majority of tissue CD8+ T cells in rheumatoid arthritis (RA) synovium, inflammatory bowel disease and other inflamed organs express granzyme K (GZMK)3, a tryptase-like protease with poorly defined function. Here, we show that GZMK can activate the complement cascade by cleaving C2 and C4. The nascent C4b and C2a fragments form a C3 convertase that cleaves C3, allowing further assembly of a C5 convertase that cleaves C5. The resulting convertases trigger every major event in the complement cascade, generating the anaphylatoxins C3a and C5a, the opsonins C4b and C3b, and the membrane attack complex. In RA synovium, GZMK is enriched in areas with abundant complement activation, and fibroblasts are the major producers of complement C2, C3, and C4 that serve as targets for GZMK-mediated complement activation. Our findings describe a previously unidentified pathway of complement activation that is entirely driven by lymphocyte-derived GZMK and proceeds independently of the classical, lectin, or alternative pathways. Given the widespread abundance of GZMK-expressing T cells in tissues in chronic inflammatory diseases and infection, GZMK-mediated complement activation is likely to be an important contributor to tissue inflammation in multiple disease contexts.
RESUMEN
Severe asthma and sinus disease are consequences of type 2 inflammation (T2I), mediated by interleukin (IL)-33 signaling through its membrane-bound receptor, ST2. Soluble (s)ST2 reduces available IL-33 and limits T2I, but little is known about its regulation. We demonstrate that prostaglandin E2 (PGE2) drives production of sST2 to limit features of lung T2I. PGE2-deficient mice display diminished sST2. In humans with severe respiratory T2I, urinary PGE2 metabolites correlate with serum sST2. In mice, PGE2 enhanced sST2 secretion by mast cells (MCs). Mice lacking MCs, ST2 expression by MCs, or E prostanoid (EP)2 receptors by MCs showed reduced sST2 lung concentrations and strong T2I. Recombinant sST2 reduced T2I in mice lacking PGE2 or ST2 expression by MCs back to control levels. PGE2 deficiency also reversed the hyperinflammatory phenotype in mice lacking ST2 expression by MCs. PGE2 thus suppresses T2I through MC-derived sST2, explaining the severe T2I observed in low PGE2 states.
Asunto(s)
Dinoprostona , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Pulmón , Mastocitos , Ratones Noqueados , Animales , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1/genética , Mastocitos/inmunología , Mastocitos/metabolismo , Dinoprostona/metabolismo , Ratones , Interleucina-33/metabolismo , Humanos , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Asma/inmunología , Asma/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Ratones Endogámicos C57BL , Inflamación/inmunología , Femenino , Masculino , Transducción de Señal , Neumonía/inmunología , Neumonía/metabolismoRESUMEN
BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is a severe disease involving dysregulated type 2 inflammation. However, the role other inflammatory pathways play in AERD is poorly understood. OBJECTIVE: We sought to broadly define the inflammatory milieu of the upper respiratory tract in AERD and to determine the effects of IL-4Rα inhibition on mediators of nasal inflammation. METHODS: Twenty-two AERD patients treated with dupilumab for 3 months were followed over 3 visits and compared to 10 healthy controls. Nasal fluid was assessed for 45 cytokines and chemokines using Olink Target 48. Blood neutrophils and cultured human mast cells, monocytes/macrophages, and nasal fibroblasts were assessed for response to IL-4/13 stimulation in vitro. RESULTS: Of the nasal fluid cytokines measured, nearly one third were higher in AERD patients compared to healthy controls, including IL-6 and the IL-6 family-related cytokine oncostatin M (OSM), both of which correlated with nasal albumin levels, a marker of epithelial barrier dysregulation. Dupilumab significantly decreased many nasal mediators, including OSM and IL-6. IL-4 stimulation induced OSM production from mast cells and macrophages but not from neutrophils, and OSM and IL-13 stimulation induced IL-6 production from nasal fibroblasts. CONCLUSION: In addition to type 2 inflammation, innate and IL-6-related cytokines are also elevated in the respiratory tract in AERD. Both OSM and IL-6 are locally produced in nasal polyps and likely promote pathology by negatively affecting epithelial barrier function. IL-4Rα blockade, although seemingly directed at type 2 inflammation, also decreases mediators of innate inflammation and epithelial dysregulation, which may contribute to dupilumab's therapeutic efficacy in AERD.
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Anticuerpos Monoclonales Humanizados , Asma Inducida por Aspirina , Subunidad alfa del Receptor de Interleucina-4 , Interleucina-6 , Oncostatina M , Transducción de Señal , Humanos , Oncostatina M/metabolismo , Femenino , Masculino , Persona de Mediana Edad , Interleucina-6/metabolismo , Interleucina-6/inmunología , Adulto , Subunidad alfa del Receptor de Interleucina-4/metabolismo , Subunidad alfa del Receptor de Interleucina-4/inmunología , Asma Inducida por Aspirina/inmunología , Mastocitos/inmunología , Mastocitos/metabolismo , Células Cultivadas , Anciano , Fibroblastos/metabolismo , Fibroblastos/inmunología , Mucosa Nasal/inmunología , Mucosa Nasal/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismoRESUMEN
Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.
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Factor Nuclear 1-alfa del Hepatocito , Hipersensibilidad , Factor de Unión 1 al Potenciador Linfoide , Células Madre Multipotentes , Factor 1 de Transcripción de Linfocitos T , Células Th2 , Humanos , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Factor de Unión 1 al Potenciador Linfoide/genética , Células Th2/inmunología , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 1-alfa del Hepatocito/genética , Hipersensibilidad/inmunología , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Diferenciación Celular , Citocinas/metabolismo , Linfopoyetina del Estroma Tímico , Animales , Células Cultivadas , RatonesRESUMEN
Macrophages expressing group V phospholipase A2 (Pla2g5) release the free fatty acid (FFA) linoleic acid (LA), potentiating lung type 2 inflammation. Although Pla2g5 and LA increase in viral infections, their role remains obscure. We generated Pla2g5flox/flox mice, deleted Pla2g5 by using the Cx3cr1cre transgene, and activated bone marrow-derived macrophages (BM-Macs) with poly:IC, a synthetic double-stranded RNA that triggers a viral-like immune response, known Pla2g5-dependent stimuli (IL-4, LPS + IFNγ, IL-33 + IL-4 + GM-CSF) and poly:IC + LA followed by lipidomic and transcriptomic analysis. Poly:IC-activated Pla2g5flox/flox;Cx3cr1cre/+ BM-Macs had downregulation of major bioactive lipids and critical enzymes producing those bioactive lipids. In addition, AKT phosphorylation was lower in poly:IC-stimulated Pla2g5flox/flox;Cx3cr1cre/+ BM-Macs, which was not restored by adding LA to poly:IC-stimulated BM-Macs. Consistently, Pla2g5flox/flox;Cx3cr1cre/+ mice had diminished poly:IC-induced lung inflammation, including inflammatory macrophage proliferation, while challenging Pla2g5flox/flox;Cx3cr1cre/+ mice with poly:IC + LA partially restored lung inflammation and inflammatory macrophage proliferation. Finally, mice lacking FFA receptor-1 (Ffar1)-null mice had reduced poly:IC-induced lung cell recruitment and tissue macrophage proliferation, not corrected by LA. Thus, Pla2g5 contributes to poly:IC-induced lung inflammation by regulating inflammatory macrophage proliferation and LA/Ffar1-mediated lung cell recruitment and tissue macrophage proliferation.
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Ácido Linoleico , Neumonía , Animales , Ratones , Proliferación Celular , Interleucina-4/metabolismo , Ácido Linoleico/metabolismo , Pulmón , MacrófagosRESUMEN
KEY POINTS: Mast cell numbers were reduced in samples cryopreserved as whole tissue chunks. Thawed epithelial cells had reduced proliferation rates when preserved as dissociated cell suspensions. The right cryopreservation method to choose may depend on the goals and cell-type focus of the project.
RESUMEN
BACKGROUND: IgE-induced mast cell (MC) degranulation can be inhibited by IgG antibodies, signaling via FcγRIIb, but the effects of IgG on IgE-induced MC transcription are unknown. OBJECTIVE: We sought to assess inhibitory IgG:FcγRIIb effects on MC responses to IgE using complementary transcriptomic and functional approaches. METHODS: RNA sequencing was performed on bone marrow-derived MCs from wild-type and FcγRIIb-deficient mice to identify genes activated following IgE receptor crosslinking that were further modulated in the presence of antigen-specific IgG in an FcγRIIb-dependent fashion. Parallel analyses of signaling pathways and allergic responses in vivo were performed to assess the impact of these changes in gene expression. RESULTS: Rapid changes in the transcription of 879 genes occurred in MCs activated by IgE, peaking at 1 hour. Surprisingly, only 12% of these were altered by IgG signaling via FcγRIIb, including numerous transcripts involved in orchestrating type 2 responses linked to spleen tyrosine kinase signaling. Consistent with this finding, IgG suppressed IgE-induced phospho-intermediates in the spleen tyrosine kinase signaling pathway. In vivo studies confirmed that the IgG-mediated suppression of both systemic anaphylaxis and MC-driven tissue recruitment of inflammatory cells following allergen challenge was dependent on FcγRIIb. In contrast, genes in the STAT5a cell survival pathway were unaltered by IgG, and STAT5a phosphorylation increased after IgE-induced MC activation but was unaffected by IgG. CONCLUSIONS: Our findings indicate that inhibitory IgG:FcγRIIb signals block an IgE-induced proallergic program but spare a prosurvival program.
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Anafilaxia , Receptores de IgE , Ratones , Animales , Receptores de IgG , Quinasa Syk/metabolismo , Inmunoglobulina E , Mastocitos , Inmunoglobulina G , Degranulación de la CélulaRESUMEN
Mast cells (MCs) are widely recognized as central effector cells during type 2 inflammatory reactions and thought to also play a role in innate immune responses, wound healing, and potentially cancer. Circulating progenitor cells mature to MCs in peripheral tissues, where they exhibit phenotypic and functional heterogeneity. This diversity likely originates from differences in MC development imprinted by microenvironmental signals. The advent of single-cell transcriptomics reveals MC diversity beyond differences in proteases that were classically used to identify MC phenotypes. Here, we provide an overview of the current knowledge on MC progenitor differentiation and characteristics, and MC heterogeneity seen in health versus disease, that are drastically advanced through single-cell profiling technologies. This powerful approach can provide detailed cellular maps of tissues to decipher the complex cellular functions and interactions that may lead to identifying candidate factors to target in therapies.
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Hipersensibilidad , Transcriptoma , Diferenciación Celular , Humanos , Hipersensibilidad/metabolismo , Mastocitos/metabolismo , Péptido Hidrolasas/metabolismo , Células MadreRESUMEN
Mast cells are heterogeneous, tissue-resident immune effector cells widely recognized to play pathobiologic roles in the development of mucosal type 2 inflammation. While mast cell progenitors can be found in peripheral blood, phenotypically mature mast cells can only be found within peripheral tissues. Here, we describe optimized tissue digestion protocols for obtaining mast cells from the murine lung and from human sinus tissue. Following tissue digestion, mast cells can be identified and sorted by flow cytometry using antibodies against defined surface markers. We also provide protocol for intracellular protease immunostaining, allowing for further characterization of mast cells.
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Pulmón , Mastocitos , Animales , Citometría de Flujo , Humanos , Mastocitos/metabolismo , Ratones , Péptido Hidrolasas/metabolismo , Células MadreRESUMEN
BACKGROUND: Mast cells (MCs) are pleiotropic cells that accumulate in the esophagus of patients with eosinophilic esophagitis (EoE) and are thought to contribute to disease pathogenesis, yet their properties and functions in this organ are largely unknown. OBJECTIVES: This study aimed to perform a comprehensive molecular and spatial characterization of esophageal MCs in EoE. METHODS: Esophageal biopsies obtained from patients with active EoE, patients with EoE in histologic remission, and individuals with histologically normal esophageal biopsies and no history of esophageal disease (ie, control individuals) were subject to single-cell RNA sequencing, flow cytometry, and immunofluorescence analyses. RESULTS: This study probed 39,562 single esophageal cells by single-cell RNA sequencing; approximately 5% of these cells were MCs. Dynamic MC expansion was identified across disease states. During homeostasis, TPSAB1highAREGhigh resident MCs were mainly detected in the lamina propria and exhibited a quiescent phenotype. In patients with active EoE, resident MCs assumed an activated phenotype, and 2 additional proinflammatory MC populations emerged in the intraepithelial compartment, each linked to a proliferating MKI67high cluster. One proinflammatory activated MC population, marked as KIThighIL1RL1highFCER1Alow, was not detected in disease remission (termed "transient MC"), whereas the other population, marked as CMA1highCTSGhigh, was detected in disease remission where it maintained an activated state (termed "persistent MC"). MCs were prominent producers of esophageal IL-13 mRNA and protein, a key therapeutic target in EoE. CONCLUSIONS: Esophageal MCs comprise heterogeneous populations with transcriptional signatures associated with distinct spatial compartmentalization and EoE disease status. In active EoE, they assume a proinflammatory state and locally proliferate, and they remain activated and poised to reinitiate inflammation even during disease remission.
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Esofagitis Eosinofílica , Proliferación Celular , Esofagitis Eosinofílica/genética , Esofagitis Eosinofílica/metabolismo , Humanos , Mastocitos/patología , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: Eosinophilic asthma and nasal polyposis are hallmarks of aspirin-exacerbated respiratory disease (AERD), and IL-5 inhibition has been shown to provide therapeutic benefit. However, IL-5Rα is expressed on many cells in addition to eosinophils, and the mechanisms by which IL-5 inhibition leads to clinical benefit in eosinophilic asthma and nasal polyposis are unlikely to be due exclusively to antieosinophil effects. OBJECTIVE: We sought to identify the mechanisms by which anti-IL-5 treatment with mepolizumab improves respiratory inflammation in AERD. METHODS: The clinical characteristics, circulating granulocytes, nasal scraping transcripts, eosinophilic cationic protein, tryptase, and antibody levels, and urinary and nasal eicosanoid levels were measured for 18 subjects with AERD who were taking mepolizumab and compared with those of 18 matched subjects with AERD who were not taking mepolizumab. RESULTS: Subjects taking mepolizumab had significantly fewer peripheral blood eosinophils and basophils, and those cells that remained had higher surface CRTH2 expression than did the cells from subjects not taking mepolizumab. Nasal prostaglandin F2α, prostaglandin D2 metabolites, leukotriene B4, and thromboxane levels were lower in subjects taking mepolizumab, as were urinary levels of tetranor-prostaglandin D2 and leukotriene E4. The nasal epithelial cell transcripts that were overexpressed among subjects with AERD who were taking mepolizumab were enriched for genes involved in tight junction formation and cilium organization. Nasal and urinary prostaglandin E2, tryptase, and antibody levels were not different between the 2 groups. CONCLUSION: IL-5 inhibition in AERD decreases production of inflammatory eicosanoids and upregulates tight junction-associated nasal epithelial cell transcripts, likely due to decreased IL-5 signaling on tissue mast cells, eosinophils, and epithelial cells. These direct effects on multiple relevant immune cells contribute to the mechanism of benefit afforded by mepolizumab.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Asma Inducida por Aspirina , Basófilos , Eosinófilos , Pólipos Nasales , Adolescente , Adulto , Anciano , Asma Inducida por Aspirina/tratamiento farmacológico , Asma Inducida por Aspirina/inmunología , Asma Inducida por Aspirina/orina , Basófilos/inmunología , Basófilos/patología , Eicosanoides/inmunología , Eicosanoides/orina , Eosinófilos/inmunología , Eosinófilos/patología , Femenino , Humanos , Interleucina-5/inmunología , Subunidad alfa del Receptor de Interleucina-5/inmunología , Masculino , Persona de Mediana Edad , Pólipos Nasales/tratamiento farmacológico , Pólipos Nasales/inmunología , Pólipos Nasales/orinaAsunto(s)
Inmunoglobulina E , Mastocitos , Recuento de Células , Liberación de Histamina , Humanos , LactanteRESUMEN
The American Initiative in Mast Cell Diseases (AIM) held its inaugural investigator conference at Stanford University School of Medicine in May 2019. The overarching goal of this meeting was to establish a Pan-American organization of physicians and scientists with multidisciplinary expertise in mast cell disease. To serve this unmet need, AIM envisions a network where basic, translational, and clinical researchers could establish collaborations with both academia and biopharma to support the development of new diagnostic methods, enhanced understanding of the biology of mast cells in human health and disease, and the testing of novel therapies. In these AIM proceedings, we highlight selected topics relevant to mast cell biology and provide updates regarding the recently described hereditary alpha-tryptasemia. In addition, we discuss the evaluation and treatment of mast cell activation (syndromes), allergy and anaphylaxis in mast cell disorders, and the clinical and biologic heterogeneity of the more indolent forms of mastocytosis. Because mast cell disorders are relatively rare, AIM hopes to achieve a coordination of scientific efforts not only in the Americas but also in Europe by collaborating with the well-established European Competence Network on Mastocytosis.
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Mastocitosis/diagnóstico , Mastocitosis/etiología , Mastocitosis/terapia , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Humanos , Mastocitosis/complicaciones , Investigación , Investigación Biomédica TraslacionalRESUMEN
Mast cells (MCs) play a pathobiologic role in type 2 (T2) allergic inflammatory diseases of the airway, including asthma and chronic rhinosinusitis with nasal polyposis (CRSwNP). Distinct MC subsets infiltrate the airway mucosa in T2 disease, including subepithelial MCs expressing the proteases tryptase and chymase (MCTC) and epithelial MCs expressing tryptase without chymase (MCT). However, mechanisms underlying MC expansion and the transcriptional programs underlying their heterogeneity are poorly understood. Here, we use flow cytometry and single-cell RNA-sequencing (scRNA-seq) to conduct a comprehensive analysis of human MC hyperplasia in CRSwNP, a T2 cytokine-mediated inflammatory disease. We link discrete cell surface phenotypes to the distinct transcriptomes of CRSwNP MCT and MCTC, which represent polarized ends of a transcriptional gradient of nasal polyp MCs. We find a subepithelial population of CD38highCD117high MCs that is markedly expanded during T2 inflammation. These CD38highCD117high MCs exhibit an intermediate phenotype relative to the expanded MCT and MCTC subsets. CD38highCD117high MCs are distinct from circulating MC progenitors and are enriched for proliferation, which is markedly increased in CRSwNP patients with aspirin-exacerbated respiratory disease, a severe disease subset characterized by increased MC burden and elevated MC activation. We observe that MCs expressing a polyp MCT-like effector program are also found within the lung during fibrotic diseases and asthma, and further identify marked differences between MCTC in nasal polyps and skin. These results indicate that MCs display distinct inflammation-associated effector programs and suggest that in situ MC proliferation is a major component of MC hyperplasia in human T2 inflammation.
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Mucosa Nasal/patología , Pólipos Nasales/inmunología , Rinitis/inmunología , Sinusitis/inmunología , Adulto , Anciano , Proliferación Celular , Endoscopía , Femenino , Citometría de Flujo , Humanos , Masculino , Mastocitos , Persona de Mediana Edad , Mucosa Nasal/citología , Mucosa Nasal/inmunología , Mucosa Nasal/cirugía , Pólipos Nasales/patología , Procedimientos Quírurgicos Nasales , RNA-Seq , Rinitis/patología , Rinitis/cirugía , Análisis de la Célula Individual , Sinusitis/patología , Sinusitis/cirugía , Adulto JovenRESUMEN
Murine mast cells (MCs) contain two lineages: inducible bone marrow-derived mucosal MCs (MMCs) and constitutive embryonic-derived connective tissue MCs (CTMCs). Here, we use RNA sequencing, flow cytometry, and genetic deletion in two allergic lung inflammation models to define these two lineages. We found that inducible MCs, marked by ß7 integrin expression, are highly distinct from airway CTMCs at rest and during inflammation and unaffected by targeted CTMC deletion. ß7High MCs expand and mature during lung inflammation as part of a TGF-ß-inducible transcriptional program that includes the MMC-associated proteases Mcpt1 and Mcpt2, the basophil-associated protease Mcpt8, granule components, and the epithelial-binding αE integrin. In vitro studies using bone marrow-derived MCs (BMMCs) identified a requirement for SCF in this this TGF-ß-mediated development and found that epithelial cells directly elicit TGF-ß-dependent BMMC up-regulation of mMCP-1 and αE integrin. Thus, our findings characterize the expansion of a distinct inducible MC subset in C57BL/6 mice and highlight the potential for epithelium to direct MMC development.
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Asma/inmunología , Células de la Médula Ósea/inmunología , Linaje de la Célula/inmunología , Mastocitos/inmunología , Mucosa Respiratoria/inmunología , Animales , Asma/embriología , Asma/genética , Asma/patología , Células de la Médula Ósea/patología , Linaje de la Célula/genética , Cadenas beta de Integrinas/genética , Cadenas beta de Integrinas/inmunología , Mastocitos/patología , Ratones , Ratones Transgénicos , Mucosa Respiratoria/embriología , Mucosa Respiratoria/patología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Triptasas/genética , Triptasas/inmunologíaRESUMEN
Macrophages have diverse functions in the pathogenesis, resolution, and repair of inflammatory processes. Elegant studies have elucidated the metabolomic and transcriptomic profiles of activated macrophages. However, the versatility of macrophage responses in inflammation is likely due, at least in part, to their ability to rearrange their repertoire of bioactive lipids, including fatty acids and oxylipins. This review will describe the fatty acids and oxylipins generated by macrophages and their role in type 1 and type 2 immune responses. We will highlight lipidomic studies that have shaped the current understanding of the role of lipids in macrophage polarization.