Critical role for the NLRP3 inflammasome during acute lung injury.

The inflammasome is a key factor in innate immunity and senses soluble pathogen and danger-associated molecular patterns as well as biological crystals (urate, cholesterol, etc.), resulting in expression of IL-1ß and IL-18. Using a standard model of acute lung injury (ALI) in mice featuring airway instillation of LPS, ALI was dependent on availability of NLRP3 as well as caspase-1, which are known features of the NLRP3 inflammasome. The appearance of IL-1ß, a product of NLRP3 inflammasome activation, was detected in bronchoalveolar lavage fluids (BALF) in a macrophage- and neutrophil-dependent manner. Neutrophil-derived extracellular histones appeared in the BALF during ALI and directly activated the NLRP3 inflammasome. Ab-mediated neutralization of histones significantly reduced IL-1ß levels in BALF during ALI. Inflammasome activation by extracellular histones in LPS-primed macrophages required NLRP3 and caspase-1 as well as extrusion of K(+), increased intracellular Ca(2+) concentration, and generation of reactive oxygen species. NLRP3 and caspase-1 were also required for full extracellular histone presence during ALI, suggesting a positive feedback mechanism. Extracellular histone and IL-1ß levels in BALF were also elevated in C5a-induced and IgG immune complex ALI models, suggesting a common inflammatory mechanism. These data indicate an interaction between extracellular histones and the NLRP3 inflammasome, resulting in ALI. Such findings suggest novel targets for treatment of ALI, for which there is currently no known efficacious drug.

Bidirectional Crosstalk between C5a Receptors and the NLRP3 Inflammasome in Macrophages and Monocytes.

C5a is an inflammatory mediator generated by complement activation that positively regulates various arms of immune defense, including Toll-like receptor 4 (TLR4) signaling. The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is activated by pathogen products and cellular/tissue damage products and is a major contributor of IL-1ß. In this study, we investigate whether C5a modulates lipopolysaccharide- (LPS-) induced NLRP3 inflammasome activation in myeloid cells. Appearance of plasma IL-1ß during endotoxemia was reduced in C5aR1(-/-) mice when compared to wild-type mice. In vitro, C5a significantly enhanced LPS-induced production of IL-1ß in bone marrow Ly6C-high inflammatory monocytes, accompanied by augmented intracellular pro-IL-1ß expression. This effect was abolished during p38 blockade by SB 203580 and in the absence of C5aR1. Conversely, C5a suppressed LPS-induced macrophage production of IL-1ß, which was accompanied by attenuated levels of pro-IL-1ß, NLRP3, and caspase-1 expression. C5a's suppressive effects were negated during phosphoinositide 3-kinase (PI3K) inhibition by wortmannin but were largely preserved in the absence of C5aR1. Thus, C5a bidirectionally amplifies TLR4-mediated NLRP3 inflammasome activation in monocytes while suppressing this pathway in macrophages. However, as C5aR1 deficiency attenuates the IL-1ß response to LPS challenge in vivo, our results suggest overall that C5a augments physiologic inflammasome responses.

The interaction between C5a and both C5aR and C5L2 receptors is required for production of G-CSF during acute inflammation.

The complement activation product, C5a, is a key factor for regulation of inflammatory responses. C5a and C5adesArg bind to their receptors, C5aR and C5L2, but the functional roles of C5L2 remain controversial. We screened the patterns of 23 inflammatory mediators in cultures of LPS-activated mouse peritoneal elicited macrophages (PEMs) in the presence or absence of recombinant mouse C5a. Production of most mediators studied was suppressed by C5a, whereas G-CSF production was enhanced. G-CSF gene expression and secretion from PEMs was amplified two- to threefold by C5a in a dose- and time-dependent fashion. The degradation product C5adesArg promoted lower levels of G-CSF. The effects of C5a on G-CSF were associated with activation of PI3K/Akt and MEK1/2 signaling pathways. C5a did not enhance G-CSF production in cultures of PEMs from either C5aR- or C5L2-deficient mice, indicating that both C5a receptors are indispensable for mediating the effects of C5a in the production of G-CSF. Finally, G-CSF levels in plasma during polymicrobial sepsis after cecal ligation and puncture were substantially lower in C5aR- or C5L2-deficient mice as compared with that in C57BL/6J WT mice. These findings elucidate the functional characteristics of the C5L2 receptor during the acute inflammatory response.

Regulation of IL-17 family members by adrenal hormones during experimental sepsis in mice.

Severe sepsis is a life-threatening disease that causes major morbidity and mortality. Catecholamines and glucocorticoids often have been used for the treatment of sepsis. Several recent studies have suggested a potential role of IL-17 during the development and progression of sepsis in small animal models. In this study, the cross-talk of catecholamines and glucocorticoids with members of the IL-17 family was investigated during sepsis in C57BL/6 mice. The concentrations in plasma of IL-17A, IL-17F, and the IL-17AF heterodimer all were increased greatly in mice after endotoxemia or cecal ligation and puncture as compared with sham mice. Surprisingly, when compared with IL-17A (487 pg/mL), the concentrations of IL-17F (2361 pg/mL) and the heterodimer, IL-17AF (5116 pg/mL), were much higher 12 hours after endotoxemia. After surgical removal of the adrenal glands, mice had much higher mortality after endotoxemia or cecal ligation and puncture. The absence of endogenous adrenal gland hormones (cortical and medullary) was associated with 3- to 10-fold higher concentrations of IL-17A, IL-17F, IL-17AF, and IL-23. The addition of adrenaline, noradrenaline, hydrocortisone, or dexamethasone to lipopolysaccharide-activated peritoneal macrophages dose-dependently suppressed the expression and release of IL-17s. The production of IL-17s required activation of c-Jun-N-terminal kinase, which was antagonized by both catecholamines and glucocorticoids. These data provide novel insights into the molecular mechanisms of immune modulation by catecholamines and glucocorticoids during acute inflammation.

Complement activation product C5a is a selective suppressor of TLR4-induced, but not TLR3-induced, production of IL-27(p28) from macrophages.

There is accumulating evidence that the complement activation product, C5a, can orchestrate cellular immune functions. IL-27(p28/EBI3) is an emerging key player essential for regulating inflammatory responses and T cells. In this article, we report that C5a robustly suppressed IL-27(p28) gene expression and release in peritoneal macrophages. These cells from C57BL/6J mice abundantly produced IL-27(p28) after engagement of either the TLR3 (polyinosinic-polycytidylic acid) or TLR4 (LPS) receptor. Genetic deficiency of either TLR4 or LBP completely incapacitated the ability of macrophages to secrete IL-27(p28) in response to LPS. IL-27(p28)-producing macrophages also expressed the C5aR receptor, thus displaying an IL-27(p28)(+)F4/80(+)C5aR(+) phenotype. C5a suppressed IL-27(p28) in LPS-stimulated macrophages via interactions with the C5aR receptor rather than the C5L2 receptor. After endotoxemia, C5aR(-/-) mice displayed higher plasma levels of IL-27(p28) compared with C57BL/6J mice. C5a did not affect the release of IL-27(p28) or the frequency of IL-27(p28)(+)F4/80(+) macrophages after engagement of TLR3. Mechanistically, LPS activated both the NF-κB and the PI3K/Akt pathways, whereas C5a activated only the PI3K/Akt pathway. Engagement of PI3K/Akt was inhibitory for IL-27(p28) production, because PI3K/Akt pharmacologic blockade resulted in increased amounts of IL-27(p28) and reversed the suppressive effects of C5a. Blockade of PI3K/Akt in endotoxemic C57BL/6J mice resulted in higher generation of IL-27(p28). In contrast, the PI3K/Akt pathway was not involved in TLR3-mediated release of IL-27(p28). These data provide new evidence about how complement activation may selectively interfere with production of T cell regulatory cytokines by APCs in the varying contexts of either bacterial (TLR4 pathway) or viral (TLR3 pathway) infection.

ATP citrate lyase links increases in glycolysis to diminished release of vesicular suppressor of cytokine signaling 3 by alveolar macrophages.

Extracellular vesicles (EVs) are important vectors for intercellular communication. Lung-resident alveolar macrophages (AMs) tonically secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis in the distal lung via its actions in recipient neighboring epithelial cells. AMs are metabolically distinct and exhibit low levels of glycolysis at steady state. To our knowledge, whether cellular metabolism influences the packaging and release of an EV cargo molecule has never been explored in any cellular context. Here, we report that increases in glycolysis following in vitro exposure of AMs to the growth and activating factor granulocyte-macrophage colony-stimulating factor inhibit the release of vesicular SOCS3 by primary AMs. Glycolytically diminished SOCS3 secretion requires export of citrate from the mitochondria to the cytosol and its subsequent conversion to acetyl-CoA by ATP citrate lyase. Our data for the first time implicate perturbations in intracellular metabolites in the regulation of vesicular cargo packaging and secretion.

Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3.

Extracellular vesicles (EVs) contain a diverse array of molecular cargoes that alter cellular phenotype and function following internalization by recipient cells. In the lung, alveolar macrophages (AMs) secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis via vesicular transfer to neighboring alveolar epithelial cells. Although changes in the secretion of EV molecules-including but not limited to SOCS3-have been described in response to microenvironmental stimuli, the cellular and molecular machinery that control alterations in vesicular cargo packaging remain poorly understood. Furthermore, the use of quantitative methods to assess the sorting of cytosolic cargo molecules into EVs is lacking. Here, we utilized cigarette smoke extract (CSE) exposure of AMs as an in vitro model of oxidative stress to address these gaps in knowledge. We demonstrate that the accumulation of reactive oxygen species (ROS) in AMs was sufficient to augment vesicular SOCS3 release in this model. Using nanoparticle tracking analysis (NTA) in tandem with a new carboxyfluorescein succinimidyl ester (CFSE)-based intracellular protein packaging assay, we show that the stimulatory effects of CSE were at least in part attributable to elevated amounts of SOCS3 packaged per EV secreted by AMs. Furthermore, the use of a 20S proteasome activity assay alongside treatment of AMs with conventional proteasome inhibitors strongly suggest that ROS stimulated SOCS3 release via inactivation of the proteasome. These data demonstrate that tuning of AM proteasome function by microenvironmental oxidants is a critical determinant of the packaging and secretion of cytosolic SOCS3 protein within EVs.

Microenvironmental Influences on Extracellular Vesicle-Mediated Communication in the Lung.

Extracellular vesicles (EVs) are increasingly appreciated as important vectors of information transmission between cells. Most research on EVs has emphasized their roles in inflammatory and pathologic conditions, and in the airways and alveoli, EV secretion by various cell types is implicated in various forms of lung disease. However, recent evidence also demonstrates a homeostatic role for lung EVs by mediating transmission of anti-inflammatory signals between alveolar macrophages and lung epithelial cells. Nevertheless, our understanding of the mechanisms responsible for EV cargo packaging, stability in the extracellular milieu, and acquisition by recipient cells remains limited. Here, we review the current understanding of these mechanistic aspects of vesicular communication and their potential modulation by constituents of the unique microenvironment of the lung.

Transcription factor Dlx3 induces aryl hydrocarbon receptor promoter activity.

The Distal-less (Dlx) homeobox transcription factors (TFs) play a prominent role in regulating multiple facets of vertebrate biology. Though widely studied as mediators of tissue development, recent work has uncovered a role for this TF family in modulating the vertebrate hematopoietic compartment. Pertinent to our study, murine Dlx1-3 are expressed in an innate lymphocyte population known as natural killer (NK) cells, and they are implicated to assume a functional role in the NK cell maturation pathway. However, Dlx target genes are poorly understood. In Drosophila, the invertebrate Dlx ortholog Distal-less (Dll) regulates another transcription factor called Spineless (ss), which is critical for specifying distal antennal segments. Importantly, the vertebrate ortholog of ss is the aryl hydrocarbon receptor (AhR), a transcription factor recently shown to be important in the regulation of a number of immune cell subsets, including NK cells. Given these findings, we investigated whether Dlx TF family members might analogously regulate AhR in an NK cell context. Our results demonstrate that Dlx3 is constitutively co-expressed with AhR in murine and human CD127+ NK cells. Critically, we show that Dlx3 induces AhR promoter activity by binding to a regulatory region that resides ~5.5 kb upstream of the transcriptional start site. This mechanism is functionally relevant, as Dlx3 expression in human NK cells significantly enhances TF activity at AhR DNA-binding elements (Xenobiotic Responsive Elements, XREs). Thus, our study defines Dlx3 as a positive regulator of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor is required for the maintenance of liver-resident natural killer cells.

A tissue-resident population of natural killer cells (NK cells) in the liver has recently been described to have the unique capacity to confer immunological memory in the form of hapten-specific contact hypersensitivity independent of T and B cells. Factors regulating the development and maintenance of these liver-resident NK cells are poorly understood. The aryl hydrocarbon receptor (AhR) is a transcription factor modulated by exogenous and endogenous ligands that is important in the homeostasis of immune cells at barrier sites, such as the skin and gut. In this study, we show that liver-resident NK (NK1.1+CD3-) cells, defined as CD49a+TRAIL+CXCR6+DX5- cells in the mouse liver, constitutively express AhR. In AhR-/- mice, there is a significant reduction in the proportion and absolute number of these cells, which results from a cell-intrinsic dependence on AhR. This deficiency in liver-resident NK cells appears to be the result of higher turnover and increased susceptibility to cytokine-induced cell death. Finally, we show that this deficiency has functional implications in vivo. Upon hapten exposure, AhR-/- mice are not able to mount an NK cell memory response to hapten rechallenge. Together, these data demonstrate the requirement of AhR for the maintenance of CD49a+TRAIL+CXCR6+DX5- liver-resident NK cells and their hapten memory function.

NKG2A-Expressing Natural Killer Cells Dominate the Response to Autologous Lymphoblastoid Cells Infected with Epstein-Barr Virus.

Epstein-Barr virus (EBV) is a human γ-herpesvirus that establishes latency and lifelong infection in host B cells while achieving a balance with the host immune response. When the immune system is perturbed through immunosuppression or immunodeficiency, however, these latently infected B cells can give rise to aggressive B cell lymphomas. Natural killer (NK) cells are regarded as critical in the early immune response to viral infection, but their role in controlling expansion of infected B cells is not understood. Here, we report that NK cells from healthy human donors display increased killing of autologous B lymphoblastoid cell lines (LCLs) harboring latent EBV compared to primary B cells. Coculture of NK cells with autologous EBV+ LCL identifies an NK cell population that produces IFNγ and mobilizes the cytotoxic granule protein CD107a. Multi-parameter flow cytometry and Boolean analysis reveal that these functional cells are enriched for expression of the NK cell receptor NKG2A. Further, NKG2A+ NK cells more efficiently lyse autologous LCL than do NKG2A- NK cells. More specifically, NKG2A+2B4+CD16-CD57-NKG2C-NKG2D+ cells constitute the predominant NK cell population that responds to latently infected autologous EBV+ B cells. Thus, a subset of NK cells is enhanced for the ability to recognize and eliminate autologous, EBV-infected transformed cells, laying the groundwork for harnessing this subset for therapeutic use in EBV+ malignancies.

Induction of M2 regulatory macrophages through the ß2-adrenergic receptor with protection during endotoxemia and acute lung injury.

The main drivers of acute inflammation are macrophages, which are known to have receptors for catecholamines. Based on their function, macrophages are broadly categorized as having either M1 (proinflammatory) or M2 phenotypes (anti-inflammatory). In this study, we investigated catecholamine-induced alterations in the phenotype of activated macrophages. In the presence of lipopolysaccharide (LPS), mouse peritoneal macrophages acquired an M1 phenotype. However, the copresence of LPS and either epinephrine or norepinephrine resulted in a strong M2 phenotype including high levels of arginase-1 and interleukin-10, and a reduced expression of M1 markers. Furthermore, epinephrine enhanced macrophage phagocytosis and promoted type 2 T-cell responses in vitro, which are known features of M2 macrophages. Analysis of M2 subtype-specific markers indicated that LPS and catecholamine-cotreated macrophages were not alternatively activated but were rather of the regulatory macrophage subtype. Interestingly, catecholamines signaled through the ß2-adrenergic receptor but not the canonical cAMP/protein kinase A signaling pathway. Instead, the M2 pathway required an intact phosphoinositol 3-kinase pathway. Blockade of the ß2-adrenergic receptor reduced survival and enhanced injury in mouse models of endotoxemia and LPS-induced acute lung injury, respectively. These results demonstrate a role for the ß2-adrenergic receptor in promoting the M2 macrophage phenotype.