Upregulated PD-L1 delays human neutrophil apoptosis and promotes lung injury in an experimental mouse model of sepsis.

PD-L1 is a ligand for PD-1, and its expression has been shown to be upregulated in neutrophils harvested from septic patients. However, the effect of PD-L1 on neutrophil survival and sepsis-induced lung injury remains largely unknown. In this study, PD-L1 expression correlated negatively with rates of apoptosis in human neutrophils harvested from patients with sepsis. Coimmunoprecipitation assays on control neutrophils challenged with interferon-γ and LPS showed that PD-L1 complexes with the p85 subunit of phosphatidyl 3-kinase (PI3K) to activate AKT-dependent survival signaling. Conditional CRE/LoxP deletion of neutrophil PD-L1 in vivo further protected against lung injury and reduced neutrophil lung infiltration in a cecal ligation and puncture (CLP) experimental sepsis animal model. Compared with wild-type animals, PD-L1-deficient animals presented lower levels of plasma tumor necrosis factor-α and interleukin-6 (IL-6) and higher levels of IL-10 after CLP, and reduced 7-day mortality in CLP PD-L1-knockout animals. Taken together, our data suggest that increased PD-L1 expression on human neutrophils delays cellular apoptosis by triggering PI3K-dependent AKT phosphorylation to drive lung injury and increase mortality during clinical and experimental sepsis.

Pre-B cell colony enhancing factor induces Nampt-dependent translocation of the insulin receptor out of lipid microdomains in A549 lung epithelial cells.

Pre-B cell colony-enhancing factor (PBEF) is a highly conserved pleiotropic protein reported to be an alternate ligand for the insulin receptor (IR). We sought to clarify the relationship between PBEF and insulin signaling by evaluating the effects of PBEF on the localization of the IRß chain to lipid rafts in A549 epithelial cells. We isolated lipid rafts from A549 cells and detected the IR by immunoprecipitation from raft fractions or whole cell lysates. Cells were treated with rPBEF, its enzymatic product nicotinamide adenine dinucleotide (NAD), or the Nampt inhibitor daporinad to study the effect of PBEF on IRß movement. We used coimmunoprecipitation studies in cells transfected with PBEF and IRß constructs to detect interactions between PBEF, the IRß, and caveolin-1 (Cav-1). PBEF was present in both lipid raft and nonraft fractions, whereas the IR was found only in lipid raft fractions of resting A549 cells. The IR-, PBEF-, and Cav-1-coimmunoprecipitated rPBEF treatment resulted in the movement of IRß- and tyrosine-phosphorylated Cav-1 from lipid rafts to nonrafts, an effect that could be blocked by daporinad, suggesting that this effect was facilitated by the Nampt activity of PBEF. The addition of PBEF to insulin-treated cells resulted in reduced Akt phosphorylation of both Ser47³ and Thr³°8. We conclude that PBEF can inhibit insulin signaling through the IR by Nampt-dependent promotion of IR translocation into the nonraft domains of A549 epithelial cells. PBEF-induced alterations in the spatial geometry of the IR provide a mechanistic explanation for insulin resistance in inflammatory states associated with upregulation of PBEF.

Activated neutrophils induce epithelial cell apoptosis through oxidant-dependent tyrosine dephosphorylation of caspase-8.

Activated neutrophils can injure host cells through direct effects of oxidants on membrane phospholipids, but an ability to induce apoptotic cell death has not previously been reported. We show that neutrophils activated in vivo in patients who have sustained multiple trauma or in vitro by exposure to bacterial lipopolysaccharide promote epithelial cell apoptosis through SHP-1-mediated dephosphorylation of epithelial cell caspase-8. Epithelial cell apoptosis induced by circulating neutrophils from patients who had sustained serious injury depended on the generation of neutrophil-derived reactive oxygen intermediates and was blocked by inhibition of NADPH oxidase or restoration of intracellular glutathione. Caspase-8 was constitutively tyrosine phosphorylated in a panel of resting epithelial cells, but underwent SHP-1-dependent dephosphorylation in response to hydrogen peroxide, activated neutrophils, or inhibition of Src kinases. Cells transfected with a mutant caspase-8 in which tyrosine residues at Tyr397 or Tyr465 are replaced by nonphosphorylatable phenylalanine underwent accelerated apoptosis, whereas either mutation of these residues to phosphomimetic glutamic acid or transfection with the Src kinases Lyn or c-Src inhibited hydrogen peroxide-induced apoptosis. Exposure to either hydrogen peroxide or lipopolysaccharide-stimulated neutrophils increased phosphorylation and activity of the phosphatase SHP-1, increased activity of caspases 8 and 3, and accelerated epithelial cell apoptosis. These observations reveal a novel mechanism for neutrophil-mediated tissue injury through oxidant-dependent, SHP-1-mediated dephosphorylation of caspase-8 resulting in enhanced epithelial cell apoptosis.

Toll-Like Receptors, Associated Biochemical Signaling Networks, and S100 Ligands.

ABSTRACT: Host cells recognize molecules that signal danger using pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are the most studied class of PRRs and detect pathogen-associated molecular patterns and danger-associated molecular patterns. Cellular TLR activation and signal transduction can therefore contain, combat, and clear danger by enabling appropriate gene transcription. Here, we review the expression, regulation, and function of different TLRs, with an emphasis on TLR-4, and how TLR adaptor protein binding directs intracellular signaling resulting in activation or termination of an innate immune response. Finally, we highlight the recent progress of research on the involvement of S100 proteins as ligands for TLR-4 in inflammatory disease.

Interleukin-1beta mediates LPS-induced inhibition of apoptosis in retinoic acid-differentiated HL-60 cells.

Promyelocytic HL-60 cells differentiated to a neutrophilic phenotype by incubation with all-trans retinoic acid become constitutively apoptotic. Exposure to either LPS or IL-1beta inhibited the apoptosis of differentiated HL-60 cells. LPS induced the expression of pro-IL-1beta message, upregulated the activity of the interleukin-1beta converting enzyme (caspase-1), and increased the release of IL-1beta into the culture medium. Prevention of IL-1beta translation with an anti-sense oligonucleotide, or prevention of IL-1beta cellular binding with a blocking antibody, accelerated rates of spontaneous apoptosis, and abrogated the inhibitory effects of LPS. However inhibition of caspase-1 activity further inhibited constitutive apoptosis of mature HL-60 cells. These studies provide further evidence of a complex regulatory pathway that modulates the expression of granulocyte apoptosis during inflammation, and point to a specific role for IL-1beta as an autocrine survival factor.

Heat-shock protein-90 prolongs septic neutrophil survival by protecting c-Src kinase and caspase-8 from proteasomal degradation.

The brief lifespan of the polymorphonuclear neutrophil (PMN) is regulated through its capacity to undergo apoptosis, a constitutive process that is actively inhibited during sepsis. We sought to define the cellular mechanisms through which Heat Shock Protein 90 (Hsp90) prolongs the survival of inflammatory PMN. We evaluated Hsp90 expression and interaction with client proteins in PMNs from patients with sepsis and in healthy control PMNs treated with LPS (1 µg/mL). Hsp90 activity was inhibited pharmacologically using radicicol (Rad; 1 µM), and Hsp90 transcription was silenced in septic PMN using siRNA. PMN apoptosis was evaluated by flow cytometry and expression of cleaved caspase-8 and -3. Septic PMNs showed reduced rates of apoptosis compared with control PMNs 21 h after isolation, and Hsp90-α mRNA was significantly more abundant in septic PMN. Caspase-8 coimmunoprecipitated with Hsp90, c-Src, and the p85 inhibitory subunit of PI3K in both septic and LPS-treated PMN. Inhibition of Hsp90 activity with Rad or its translation using siRNA restored basal rates of apoptosis in both septic and LPS-treated PMN. Radicicol further reduced c-Src protein abundance, increased the ubiquitination of caspase-8 and c-Src, and enhanced the cleavage of caspase-8 and -3. We conclude that Hsp90 prolongs the survival of activated neutrophils by stabilizing a molecular complex of c-Src kinase and caspase-8, preventing their ubiquitination, and resulting in inhibition of the catalytic activity of caspase-8 and -3.

Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis.

Pre-B cell colony-enhancing factor (PBEF) is a highly conserved 52-kDa protein, originally identified as a growth factor for early stage B cells. We show here that PBEF is also upregulated in neutrophils by IL-1beta and functions as a novel inhibitor of apoptosis in response to a variety of inflammatory stimuli. Induction of PBEF occurs 5-10 hours after LPS exposure. Prevention of PBEF translation with an antisense oligonucleotide completely abrogates the inhibitory effects of LPS, IL-1, GM-CSF, IL-8, and TNF-alpha on neutrophil apoptosis. Immunoreactive PBEF is detectable in culture supernatants from LPS-stimulated neutrophils, and a recombinant PBEF fusion protein inhibits neutrophil apoptosis. PBEF is also expressed in neutrophils from critically ill patients with sepsis in whom rates of apoptosis are profoundly delayed. Expression occurs at higher levels than those seen in experimental inflammation, and a PBEF antisense oligonucleotide significantly restores the normal kinetics of apoptosis in septic polymorphonuclear neutrophils. Inhibition of apoptosis by PBEF is associated with reduced activity of caspases-8 and -3, but not caspase-9. These data identify PBEF as a novel inflammatory cytokine that plays a requisite role in the delayed neutrophil apoptosis of clinical and experimental sepsis.

Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src.

Src family tyrosine kinases (SFKs) phosphorylate caspase-8A at tyrosine (Y) 397 resulting in suppression of apoptosis. In addition, the phosphorylation of caspase-8A at other sites including Y465 has been implicated in the regulation of caspase-8 activity. However, the functional consequences of these modifications on caspase-8 processing/activity have not been elucidated. Moreover, various Src substrates are known to act as potent Src regulators, but no such role has been explored for caspase-8. We asked whether the newly identified caspase-8 phosphorylation sites might regulate caspase-8 activation and conversely, whether caspase-8 phosphorylation might affect Src activity. Here we show that Src phosphorylates caspase-8A at multiple tyrosine sites; of these, we have focused on Y397 within the linker region and Y465 within the p12 subunit of caspase-8A. We show that phosphomimetic mutation of caspase-8A at Y465 prevents its cleavage and the subsequent activation of caspase-3 and suppresses apoptosis. Furthermore, simultaneous phosphomimetic mutation of caspase-8A at Y397 and Y465 promotes the phosphorylation of c-Src at Y416 and increases c-Src activity. Finally, we demonstrate that caspase-8 activity prevents its own tyrosine phosphorylation by Src. Together these data reveal that dual phosphorylation converts caspase-8 from a pro-apoptotic to a pro-survival mediator. Specifically, tyrosine phosphorylation by Src renders caspase-8 uncleavable and thereby inactive, and at the same time converts it to a Src activator. This novel dynamic interplay between Src and caspase-8 likely acts as a potent signal-integrating switch directing the cell towards apoptosis or survival.

Dynamic regulation of neutrophil survival through tyrosine phosphorylation or dephosphorylation of caspase-8.

Efficient expression of innate immunity is critically dependent upon the capacity of the neutrophil to be activated rapidly in the face of an acute threat and to involute once that threat has been eliminated. Here we report a novel mechanism regulating neutrophil survival dynamically through the tyrosine phosphorylation or dephosphorylation of caspase-8. Caspase-8 is tyrosine-phosphorylated in freshly isolated neutrophils but spontaneously dephosphorylates in culture, in association with the progression of constitutive apoptosis. Phosphorylation of caspase-8 on Tyr-310 facilitates its interaction with the Src-homology domain 2 containing tyrosine phosphatase-1 (SHP-1) and enables SHP-1 to dephosphorylate caspase-8, permitting apoptosis to proceed. The non-receptor tyrosine kinase, Lyn, can phosphorylate caspase-8 on Tyr-397 and Tyr-465, rendering it resistant to activational cleavage and inhibiting apoptosis. Exposure to lipopolysaccharide reduces SHP-1 activity and binding to caspase-8, caspase-8 activity, and rates of spontaneous apoptosis. SHP-1 activity is reduced and Lyn increased in neutrophils from patients with sepsis, in association with profoundly delayed apoptosis; inhibition of Lyn can partially reverse this delay. Thus the phosphorylation and dephosphorylation of caspase-8, mediated by Lyn and SHP-1, respectively, represents a novel, dynamic post-translational mechanism for the regulation of neutrophil apoptosis whose dysregulation contributes to persistent neutrophil survival in sepsis.

Delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity.

OBJECTIVE: The resolution of neutrophil (PMN)-mediated inflammation occurs through the apoptosis, or programmed cell death, of the neutrophil. PMN apoptosis is inhibited by a variety of inflammatory stimuli; moreover, PMN from critically ill septic patients show profoundly delayed rates of apoptosis in vitro. Since apoptosis is effected through the activity of intracellular cysteine proteases (caspases), we evaluated caspase expression and activity in neutrophils from septic patients and compared them with caspase expression and activity of resting or lipopolysaccharide-activated neutrophils from healthy volunteers. DESIGN: Prospective observational cohort study. SETTING: Tertiary level intensive care unit and associated research laboratory. SUBJECTS: Thirty-six intensive care unit patients with sepsis; ten healthy laboratory controls. INTERVENTIONS: Collection of up to 10 mL of whole blood for in vitro study of rates of apoptosis, expression and activity of caspases-1, -3, and -9, activation of nuclear factor-kappaB, and change in mitochondrial transmembrane potential. MEASUREMENTS AND MAIN RESULTS: Following 24 hrs of in vitro culture, 52 +/- 7.8% of control neutrophils, but only 29 +/- 5.4% of lipopolysaccharide-stimulated (1 microg/mL) PMN, showed nuclear changes of apoptosis. Only 6.2 +/- 1.1% of neutrophils from septic patients were apoptotic after 24 hrs. Significant nuclear translocation of nuclear factor-kappaB was evident in septic PMN, and inhibition of apoptosis was partially abrogated by prevention of nuclear factor-kappaB dissociation with pyrrolidine dithiocarbamate. Caspase-3 transcription and catalytic activity were significantly reduced in both patients' and lipopolysaccharide-treated PMN; caspase-1 transcription and activity were increased by lipopolysaccharide but reduced in septic patients. In contrast, caspase-9 transcription and activity were reduced in septic patients but not in lipopolysaccharide-treated PMN. Decreased caspase-9 activity was associated with sustained maintenance of mitochondrial transmembrane potential and reduced translocation of cytochrome c from the mitochondria to the cytosol. CONCLUSIONS: Apoptosis of circulating neutrophils from patients with clinical sepsis is profoundly suppressed, through a mechanism that involves activation of nuclear factor-kappaB that is associated with reduced activity of caspases-9 and -3 and maintenance of mitochondrial transmembrane potential and that differs in important respects from the inhibitory effects seen following the exposure of healthy neutrophils to inflammatory stimuli.