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Although the study of pathogen sensing by host defense systems continues to uncover a role for inflammasome components specific to particular pathogens, gaps remain in our knowledge. After internalization, Francisella escapes from the phagosome in mononuclear cells and is thought to be detected by intracellular pathogen-response-receptors pyrin and Aim2 in human and murine models, respectively. However, it remains controversial as to the role of pyrin in detecting Francisella. Our current work aims to study the contribution of inflammasome sensor, Pyrin in regulating microparticulate caspase-1/GSDM-D activation by Francisella. Our findings suggest that NLRP3 is central to the activation/release of active caspase-1/GSDM-D encapsulated in microparticles (MP) by Francisella. We also provide evidence that this regulation is independent of pyrin, implicated in sensing cytosolic Francisella in NLRP3-/- conditions where endogenous Pyrin is present. Absence of NLRP3 completely abrogated Francisella mediated MP caspase-1/GSDM-D activation and release both before and after internalization of the pathogen. However, deletion of pyrin not only enhanced both LPS and Francisella mediated MP active caspase-1/GSDM-D release, but pyrin overexpression resulted in a reduction of inflammasome activation and release; suggesting an inhibitory role of pyrin in LPS and Francisella mediated MP responses. This NLRP3 dependence and inhibitory effect of pyrin correlated with cytokine release as well. These observations also correlated with MPs ability to induce cell death; as LPS and Francisella-induced MPs from pyrin-deficient cells were more potent than wild-type monocytes whereas, NLRP3-/- MPs failed to induce cell death. Taken together, we report that NLPR3 not only mediates Francisella induced cytokine responses, but is also critical for cytokine-independent microparticle-induced inflammasome activation and endothelial cell injury independent of pyrin., Competing Interests: The authors have declared that no competing interests exist.

Inflammasome activation is a two-step process where step one, priming, prepares the inflammasome for its subsequent activation, by step two. Classically step one can be induced by LPS priming followed by step two, high dose ATP. Furthermore, when IL-18 processing is used as the inflammasome readout, priming occurs before new protein synthesis. In this context, how intracellular pathogens such as Francisella activate the inflammasome is incompletely understood, particularly regarding the relative importance of priming versus activation steps. To better understand these events we compared Francisella strains that differ in virulence and ability to induce inflammasome activation for their relative effects on step one vs. step two. When using the rapid priming model, i.e., 30 min priming by live or heat killed Francisella strains (step 1), followed by ATP (step 2), we found no difference in IL-18 release, p20 caspase-1 release and ASC oligomerization between Francisella strains (F. novicida, F. holarctica -LVS and F. tularensis Schu S4). This priming is fast, independent of bacteria viability, internalization and phagosome escape, but requires TLR2-mediated ERK phosphorylation. In contrast to their efficient priming capacity, Francisella strains LVS and Schu S4 were impaired in inflammasome triggering compared to F. novicida. Thus, observed differences in inflammasome activation by F. novicida, LVS and Schu S4 depend not on differences in priming but rather on their propensity to trigger the primed inflammasome.

Relative to monocytes, human macrophages are deficient in their ability to process and release IL-1beta. In an effort to explain this difference, we used a model of IL-1beta processing and release that is dependent upon bacterial escape into the cytosol. Fresh human blood monocytes were compared with monocyte-derived macrophages (MDM) for their IL-1beta release in response to challenge with Francisella novicida. Although both cell types produced similar levels of IL-1beta mRNA and intracellular pro-IL-1beta, only monocytes readily released processed mature IL-1beta. Baseline mRNA expression profiling of candidate genes revealed a remarkable deficiency in the pyrin gene, MEFV, expression in MDM compared with monocytes. Immunoblots confirmed a corresponding deficit in MDM pyrin protein. To determine whether pyrin levels were responsible for the monocyte/MDM difference in mature IL-1beta release, pyrin expression was knocked down by nucleofecting small interfering RNA against pyrin into monocytes or stably transducing small interfering RNA against pyrin into the monocyte cell line, THP-1. Pyrin knockdown was associated with a significant drop in IL-1beta release in both cell types. Importantly, M-CSF treatment of MDM restored pyrin levels and IL-1beta release. Similarly, the stable expression of pyrin in PMA-stimulated THP-1-derived macrophages induces caspase-1 activation, associated with increased IL-1beta release after infection with F. novicida. In summary, intracellular pyrin levels positively regulate MDM IL-1beta responsiveness to Francisella challenge.