Jamison J. Grailer

Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury

We investigated how complement activation promotes tissue injury and organ dysfunction during acute inflammation. Three models of acute lung injury (ALI) induced by LPS, IgG immune complexes, or C5a were used in C57BL/6 mice, all models requiring availability of both C5a receptors (C5aR and C5L2) for full development of ALI. Ligation of C5aR and C5L2 with C5a triggered the appearance of histones (H3 and H4) in bronchoalveolar lavage fluid (BALF). BALF from humans with ALI contained H4 histone. Histones were absent in control BALF from healthy volunteers. In mice with ALI, in vivo neutralization of H4 with IgG antibody reduced the intensity of ALI. Neutrophil depletion in mice with ALI markedly reduced H4 presence in BALF and was highly protective. The direct lung damaging effects of extracellular histones were demonstrated by airway administration of histones into mice and rats (Sprague‐Dawley), which resulted in ALI that was C5a receptor‐independent, and associated with intense inflammation, PMN accumulation, damage/destruction of alveolar epithelial cells, together with release into lung of cytokines/chemokines. High‐resolution magnetic resonance imaging demonstrated lung damage, edema and consolidation in histone‐injured lungs. These studies confirm the destructive C5a‐dependent effects in lung linked to appearance of extracellular histones.—Bosmann, M., Grailer, J. J., Ruemmler, R., Russkamp, N. F., Zetoune, F. S., Sarma, J. V., Standiford, T. J., Ward, P. A., Extracellular histones are essential effectors of C5aR‐ and C5L2‐mediated tissue damage and inflammation in acute lung injury. FASEB J. 27, 5010–5021 (2013). www.fasebj.org

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-1β 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-1β, 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-1β 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 Ca2+ 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-1β 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.

Anti-inflammatory effects of β2 adrenergic receptor agonists in experimental acute lung injury

These studies were undertaken to extend emerging evidence that β2 adrenergic receptor (β2AR) agonists, in addition to their bronchorelaxing effects, may have broad anti‐inflammatory effects in the lung following onset of experimental acute lung injury (ALI). Young male C57BL/6 mice (25 g) developed ALI following airway deposition of bacterial LPS or IgG immune complexes in the absence or presence of appropriate stereoisomers (enantiomers) of β2AR agonists, albuterol or formoterol. Endpoints included albumin leak into lung and buildup of polymorphonuclear neutrophils and cytokines/chemokines in bronchoalveolar fluids. Both β2AR agonists suppressed lung inflammatory parameters (IC50=10‐7 M). Similar effects of β2AR agonists on mediator release were found when mouse macrophages were stimulated in vitro with LPS. The protective effects were associated with reduced activation (phosphorylation) of JNK but not of other signaling proteins. Collectively, these data suggest that β2AR agonists have broad anti‐inflammatory effects in the setting of ALI. While β2AR agonists suppress JNK activation, the extent to which this can explain the blunted lung inflammatory responses in the ALI models remains to be determined.—Bosmann, M., Grailer, J. J., Zhu, K., Matthay, M A., Vidya Sarma, J., Zetoune, F. S., Ward, P. A. Anti‐inflammatory effects of β2 adrenergic receptor agonists in experimental acute lung injury. FASEB J. 26, 2137‐2144 (2012). www.fasebj.org

Induction of M2 regulatory macrophages through the β2- 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 β2-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 β2-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 β2-adrenergic receptor in promoting the M2 macrophage phenotype.

Role of extracellular histones in the cardiomyopathy of sepsis

The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca2+]i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nacht‐, LRR‐, and PYD‐domains‐containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca2+]i, as wellas defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction.—Kalbitz, M., Grailer, J. J., Fattahi, F., Jajou, L., Herron, T. J., Campbell, K. F., Zetoune, F. S., Bosmann, M., Sarma, J. V., Huber‐Lang, M., Gebhard, F., Loaiza, R., Valdivia, H. H., Jalife, J., Russell, M. W., Ward, P. A. Role of extracellular histones in the cardiomyopathy of sepsis. FASEB J. 29, 2185‐2193 (2015). www.fasebj.org

Inhibition of junctional adhesion molecule-A/LFA interaction attenuates leukocyte trafficking and inflammation in brain ischemia/reperfusion injury

Proinflammatory mediators trigger intensive postischemic inflammatory remodeling of the blood-brain barrier (BBB) including extensive brain endothelial cell surface and junctional complex changes. Junctional adhesion molecule-A (JAM-A) is a component of the brain endothelial junctional complex with dual roles: paracellular route occlusion and regulating leukocyte docking and migration. The current study examined the contribution of JAM-A to the regulation of leukocyte (neutrophils and monocytes/macrophages) infiltration and the postischemic inflammatory response in brain ischemia/reperfusion (I/R injury). Brain I/R injury was induced by transient middle cerebral artery occlusion (MCAO) for 30min in mice followed by reperfusion for 0-5days, during which time JAM-A antagonist peptide (JAM-Ap) was administered. The peptide, which inhibits JAM-A/leukocyte interaction by blocking the interaction of the C2 domain of JAM-A with LFA on neutrophils and monocytes/macrophages, attenuated I/R-induced neutrophil and monocyte infiltration into brain parenchyma. Consequently, mice treated with JAM-A peptide during reperfusion had reduced expression (~3-fold) of inflammatory mediators in the ischemic penumbra, reduced infarct size (94±39 vs 211±38mm3) and significantly improved neurological score. BBB hyperpermeability was also reduced. Collectively, these results indicate that JAM-A has a prominent role in regulating leukocyte infiltration after brain I/R injury and could be a new target in limiting post-ischemic inflammation.

Melatonin alleviates acute lung injury through inhibiting the NLRP3 inflammasome

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinically severe respiratory disorders, and there are currently no Food and Drug Administration–approved drug therapies. Melatonin is a well‐known anti‐inflammatory molecule, which has proven to be effective in ALI induced by many conditions. Emerging studies suggest that the NLRP3 inflammasome plays a critical role during ALI. How melatonin directly blocks activation of the NLRP3 inflammasome in ALI remains unclear. In this study, using an LPS‐induced ALI mouse model, we found intratracheal (i.t.) administration of melatonin markedly reduced the pulmonary injury and decreased the infiltration of macrophages and neutrophils into lung. During ALI, the NLRP3 inflammasome is significantly activated with a large amount of IL‐1β and the activated caspase‐1 occurring in the lung. Melatonin inhibits the activation of the NLRP3 inflammasome by both suppressing the release of extracellular histones and directly blocking histone‐induced NLRP3 inflammasome activation. Notably, i.t. route of melatonin administration opens a more efficient therapeutic approach for treating ALI.

Zonulin as prehaptoglobin2 regulates lung permeability and activates the complement system

Zonulin is a protein involved in the regulation of tight junctions (TJ) in epithelial or endothelial cells. Zonulin is known to affect TJ in gut epithelial cells, but little is known about its influences in other organs. Prehaptoglobin2 has been identified as zonulin and is related to serine proteases (MASPs, C1qrs) that activate the complement system. The current study focused on the role of zonulin in development of acute lung injury (ALI) in C57BL/6 male mice following intrapulmonary deposition of IgG immune complexes. A zonulin antagonist (AT-1001) and a related peptide with permeability agonist activities (AT-1002) were employed and given intratracheally or intravenously. Also, zonulin was blocked in lung with a neutralizing antibody. In a dose-dependent manner, AT-1001 or zonulin neutralizing antibody attenuated the intensity of ALI (as quantitated by albumin leak, neutrophil accumulation, and proinflammatory cytokines). A similar pattern was found using the bacterial lipopolysaccharide model of ALI. Using confocal microscopy on sections of injured lungs, staining patterns for TJ proteins were discontinuous, reduced, and fragmented. As expected, the leak of blood products into the alveolar space confirmed the passage of 3 and 20 kDa dextran, and albumin. In contrast to AT-1001, application of the zonulin agonist AT-1002 intensified ALI. Zonulin both in vitro and in vivo induced generation of complement C3a and C5a. Collectively, these data suggest that zonulin facilitates development of ALI both by enhancing albumin leak and complement activation as well as increased buildup of neutrophils and cytokines during development of ALI.

CD11c+ alveolar macrophages are a source of IL-23 during lipopolysaccharide-induced acute lung injury.

ABSTRACT Acute lung injury (ALI) is a severe pulmonary disease causing high numbers of fatalities worldwide. Innate immune responses are an integral part of the pathophysiologic events during ALI. Interleukin 23 (IL-23) is a proinflammatory mediator known to direct the inflammatory responses in various settings of infection, autoimmunity, and cancer. Interleukin 23 has been associated with proliferation and effector functions in TH17 cells. Surprisingly, little is known about production of IL-23 during ALI. In this study, we found expression of mRNA for IL-23p19 to be 10-fold elevated in lung homogenates of C57BL/6 mice after lipopolysaccharide (LPS)–induced ALI. Likewise, concentrations of IL-23 significantly increased in bronchoalveolar lavage fluids. Experiments with IL-23–deficient mice showed that endogenous IL-23 was required for production of IL-17A during LPS-ALI. CD11c-diphtheria toxin receptor transgenic mice were used to selectively deplete CD11c+ cells, the data suggesting that IL-23 production is dependent at least in part on CD11c+ cells during ALI. No alterations of IL-23 levels were observed in Rag-1–deficient mice as compared with wild-type C57BL/6 mice following ALI. The mouse alveolar macrophage cell line, MH-S, as well as primary alveolar macrophages displayed abundant surface expression of CD11c. Activation of these macrophages by LPS resulted in release of IL-23 in vitro. Our findings identify CD11c+ macrophages in the lung are likely an important source of IL-23 during ALI, which may be helpful for better understanding of this disease.

Regulatory effects of C5a on IL-17A, IL-17F, and IL-23

The complement anaphylatoxin, C5a, through binding to its receptors (C5aR or C5L2), has important biological properties for recruitment and activation of phagocytes. C5a has been identified as a powerful modulator of Toll-like receptor-induced cytokine and chemokine production by macrophages. Both the complement system and the interleukin (IL)-17 cytokine family protect against extracellular pathogens by enhancing innate immune functions. On the basis of its concentration, C5a can either positively or negatively modulate the production by macrophages of IL-17 family members as well as IL-23 via the phosphatidylinositol 3-kinase/Akt signaling cascade. C5a can also affect the production and maintenance of IL-17-producing T cells. Using C5a, C5aR, or C5L2 deficiency or blockade, IL-17/IL-23 production and/or IL-17-dependent disease progression has been shown to be substantially modified. The contributions of C5a interaction with its receptors in the production of IL-17/IL-23 and promotion of IL-17-dependent immune responses are reviewed.