Distinct mechanisms of type 3 secretion system recognition control LTB4 synthesis in neutrophils and macrophages.

Leukotriene B4 (LTB4) is an inflammatory lipid produced in response to pathogens that is critical for initiating the inflammatory cascade needed to control infection. However, during plague, Yersinia pestis inhibits the timely synthesis of LTB4 and subsequent inflammation. Using bacterial mutants, we previously determined that Y. pestis inhibits LTB4 synthesis via the action of the Yop effector proteins that are directly secreted into host cells through a type 3 secretion system (T3SS). Here, we show that the T3SS is the primary pathogen associated molecular pattern (PAMP) required for production of LTB4 in response to both Yersinia and Salmonella. However, we also unexpectantly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require the activation of two distinctly different host signaling pathways. We identified that phagocytosis and the NLRP3/CASP1 inflammasome significantly impact LTB4 synthesis by macrophages but not neutrophils. Instead, the SKAP2/PLC signaling pathway is required for T3SS-mediated LTB4 production by neutrophils. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered that a second unrelated PAMP-mediated signal activates the MAP kinase pathway needed for synthesis. Together, these data demonstrate significant differences in the host factors and signaling pathways required by macrophages and neutrophils to quickly produce LTB4 in response to bacteria. Moreover, while macrophages and neutrophils might rely on different signaling pathways for T3SS-dependent LTB4 synthesis, Y. pestis has evolved virulence mechanisms to counteract this response by either leukocyte to inhibit LTB4 synthesis and colonize the host.

In response to bacteria, neutrophils release extracellular vesicles capable of initiating thrombin generation through DNA-dependent and independent pathways.

Neutrophils release extracellular vesicles (EVs) and some subsets of neutrophil-derived EVs are procoagulant. In response to S. aureus, neutrophils produce EVs that associate electrostatically with neutrophil extracellular traps (NETs). DNA in NETs is procoagulant, but whether neutrophil EVs produced during bacterial challenge have similar activity is unknown. Given that EV activity is agonist- and cell-type dependent and coagulation contributes to sepsis, we hypothesized that sepsis-causing bacteria increase production of neutrophil-derived EVs, as well as EV-associated DNA, and intact EVs and DNA cause coagulation. We recovered EVs from neutrophils challenged with S. aureus (SA), S. epidermidis (SE), E. coli (EC), and P. aeruginosa (PA), and measured associated DNA and procoagulant activity. EVs from SA-challenged neutrophils (SA-EVs), which were previously characterized, displayed dose-dependent procoagulant activity as measured by thrombin generation (TG) in platelet-poor plasma. EV lysis and DNase treatment reduced TG by 90% and 37%, respectively. SE, EC, and PA also increased EV production and EV-associated extracellular DNA, and these EVs were also procoagulant. Compared to spontaneously released EVs, which demonstrated some ability to amplify Factor XII-dependent coagulation in the presence of an activator, only EVs produced in response to bacteria could initiate the pathway. SA-EVs and SE-EVs had more surface-associated DNA than EC-EVs and PA-EVs, and SA-EVs and SE-EVs contributed to initiation and amplification of TG in a DNA-dependent manner. However, DNA on EC- or PA-EVs played no role, suggesting that neutrophils release procoagulant EVs which can activate the coagulation cascade through both DNA-dependent and independent mechanisms.

Extracellular vesicles from A23187-treated neutrophils cause cGAS-STING-dependent IL-6 production by macrophages.

In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with S. aureus, as well as those treated with A23187, released significantly more DNA relative to untreated or fMLF-treated neutrophils, with no lysis occurring for any condition. To test the hypothesis that EVs generated during NETosis caused macrophage inflammation, we isolated and characterized EVs from A23187-treated neutrophils (A23187-EVs). A23187-EVs associated with neutrophil granule proteins, histone H3, transcription factor A, mitochondrial (TFAM), and nuclear and mitochondrial DNA (mtDNA). We showed that DNA from A23187-EVs, when transfected into macrophages, led to production of IL-6 and IFN-α2, and this response was blunted by pre-treatment with the STING inhibitor H151. Next, we confirmed that A23187-EVs were engulfed by macrophages, and showed that they induced cGAS-STING-dependent IL-6 production. In contrast, neither EVs from untreated or fMLF-treated cells exhibited pro-inflammatory activity. Although detergent-mediated lysis of A23187-EVs diminished IL-6 production, removal of surface-associated DNA with DNase I treatment had no effect, and A23187-EVs did not induce IFN-α2 production. Given these unexpected results, we investigated whether macrophage mtDNA activated the cGAS-STING signaling axis. Consistent with mitochondrial outer membrane permeabilization (MOMP), a defined mechanism of mtDNA release, we observed macrophage mitochondrial membrane depolarization, a decrease in cytosolic Bax, and a decrease in mitochondrial cytochrome c, suggesting that macrophage mtDNA may initiate this EV-dependent signaling cascade. All together, these data demonstrate that A23187-EVs behave differently than transfected NET- or EV-DNA, and that neutrophil-derived EVs could be used as a model to study NF-κB-dependent STING activation.