Efficient Neutrophil Activation Requires Two Simultaneous Activating Stimuli.

Neutrophils are abundantly present in the synovium and synovial fluid of patients suffering from arthritis. Neutrophils can be activated by a multitude of stimuli and the current dogma states that this is a two-step process, consisting of a priming step followed by an activation step. Considering that neutrophil activation occurs in an inflammatory environment, where multiple stimuli are present, we argue that a two-step process is highly unlikely. Here, we indeed demonstrate that neutrophils require simultaneous ligation of two different receptors for efficient activation. We isolated human peripheral blood neutrophils and cultured them with various combinations of stimuli (GM-CSF, fMLF, TNF, and LPS). Next, we evaluated essential neutrophil functions, including degranulation and ROS production using flow cytometry, mediator release using ELISA, NETosis by a live cell imaging method, phagocytosis by imaging flow cytometry, and extracellular vesicle (EV) release quantified by high-resolution flow cytometry. Exposure of neutrophils to any combination of stimuli, but not to single stimuli, resulted in significant degranulation, and mediator and EV release. Furthermore, ROS production increased substantially by dual stimulation, yet appeared to be more dependent on the type of stimulation than on dual stimulation. Phagocytosis was induced to its maximum capacity by a single stimulus, while NETosis was not induced by any of the used physiological stimuli. Our data indicate that neutrophil activation is tightly regulated and requires activation by two simultaneous stimuli, which is largely independent of the combination of stimuli.

Viral dsRNA-activated human dendritic cells produce IL-27, which selectively promotes cytotoxicity in naive CD8+ T cells.

Viral recognition programs DCs to express Signal 3 molecules that promote the differentiation of effector CD8(+) T cells. Besides IL-12, another DC-derived IL-12 family member, IL-27, has been reported to contribute herein, but its specific role is not well understood. Here, we show that whereas IL-12 potently induces inflammatory cytokines (i.e., IFN-γ and TNF-α, but not IL-2), IL-27 excels in inducing proliferation and a cytotoxic profile (GrB, cytotoxicity of target cells) in human naïve CD8(+) T cells. Compared with bacterial cell-wall peptidoglycan, viral dsRNA-mimic poly (I:C) is superior in priming human BDCA1(+) peripheral blood DCs to produce IL-12 and IL-27, which promote inflammatory cytokines and a cytotoxic profile in differentiating CD8(+) T cells, respectively. These data support the concept that viral dsRNA-activated human DCs produce IL-27 to act as a specialized procytotoxic, antiviral cytokine in the development of effector CD8(+) T cells.

Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells to promote interleukin-17 production in human memory T cells.

How the development of antibacterial T helper 17 (Th17) cells is selectively promoted by antigen-presenting dendritic cells (DCs) is unclear. We showed that bacteria, but not viruses, primed human DCs to promote IL-17 production in memory Th cells through the nucleotide oligomerization domain 2 (NOD2)-ligand muramyldipeptide (MDP), a derivative of bacterial peptidoglycan. MDP enhanced obligate bacterial Toll-like receptor (TLR) agonist induction of IL-23 and IL-1, which promoted IL-17 expression in T cells. The role of NOD2 in this IL-23-IL-1-IL-17 axis could be confirmed in NOD2-deficient DCs, such as DCs from selected Crohn's disease patients. Thus, antibacterial Th17-mediated immunity in humans is orchestrated by DCs upon sensing bacterial NOD2-ligand MDP.