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.

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.

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.

Modulating neutrophil apoptosis.

Polymorphonuclear neutrophils are short-lived phagocytic cells that serve as cardinal early cellular effectors of innate immunity. Both oxidative and non-oxidative mechanisms contribute to microbial killing by the neutrophil. Neutrophil defence mechanisms are potent but non-specific, with the result that inadvertent injury to host tissues commonly accompanies the activation of a neutrophil mediated response; this bystander injury has been implicated in the tissue injury of sepsis. The capacity for neutrophils to cause injury to host tissues is attenuated by the relatively short in vivo lifespan of the neutrophil, a consequence of a constitutively expressed program of apoptosis. That program can be inhibited, and neutrophil survival prolonged, through the interaction of the neutrophil with a variety of mediators of both microbial and host origin. These, in turn, inhibit apoptosis by increasing the expression of anti-apoptotic genes within the neutrophil: interleukin (IL)1beta and a novel cytokine-like molecule pre-B cell colony-enhancing factor (PBEF) are central to this inhibitory influence. Conversely, the phagocytosis of a micro-organism activates the apoptotic program, and so contributes to the resolution of acute inflammation. A complex series of interactions between the neutrophil and microorganisms or their products regulates the duration and intensity of an inflammatory response, and so provides an attractive target for therapeutic manipulation.