Closed circulatory systems (CCS) underlie the function of vertebrate organs, but in long bones their structure is unclear, although they constitute the exit route for bone marrow (BM) leukocytes. To understand neutrophil emigration from BM, we studied the vascular system of murine long bones. Here we show that hundreds of capillaries originate in BM, cross murine cortical bone perpendicularly along the shaft and connect to the periosteal circulation. Structures similar to these trans-cortical-vessels (TCVs) also exist in human limb bones. TCVs express arterial or venous markers and transport neutrophils. Furthermore, over 80% arterial and 59% venous blood passes through TCVs. Genetic and drug-mediated modulation of osteoclast count and activity leads to substantial changes in TCV numbers. In a murine model of chronic arthritic bone inflammation, new TCVs develop within weeks. Our data indicate that TCVs are a central component of the CCS in long bones and may represent an important route for immune cell export from the BM.

Highlights d Platelets license NLRP3 for inflammasome activattion in innate immune cells d Platelets are required for optimal monocyte inflammasome activation d Platelets shape IL-1b in vivo, and platelet counts correlate with IL-1b in plasma d A constitutive, heat-sensitive soluble platelet-factor boost IL-1b in macrophages

ABSTRACTThe inflammasomes control the bioactivity of pro-inflammatory cytokines of the interleukin (IL)-1 family. The inflammasome assembled by NLRP3 has been predominantly studied in homogenous cell populations in vitro, neglecting the influence of cellular interactions that occur in vivo. Here, we show that platelets, the second most abundant cells in the blood, boost the inflammasome capacity of human macrophages and neutrophils, and are critical for IL-1 production by monocytes. Platelets license NLRP3 transcription, thereby enhancing ASC nucleation, caspase-1 activity, and IL-1β maturation. Platelet depletion attenuated LPS-induced IL-1β in vivo, and platelet counts correlate with plasma concentrations of IL-1β in malaria patients. Furthermore, a platelet gene signature was enriched among the highest expressed transcripts in IL-1β-driven autoinflammatory diseases. The platelet-mediated enhancement of inflammasome activation was independent of cell-to-cell contacts, platelet-derived lipid mediators, purines, nucleic acids and a host of platelet cytokines, and involved the triggering of calcium sensing receptors on macrophages by a calcium-dependent protein commonly released by platelets and megakaryocytes. Finally, we report that platelets provide an additional layer of regulation of inflammasomes in vivo.

Inflammasomes sense intracellular clues of infection, damage, or metabolic imbalances. Activated inflammasome sensors polymerize the adaptor ASC into micron-sized "specks" to maximize caspase-1 activation and the maturation of IL-1 cytokines. Caspase-1 also drives pyroptosis, a lytic cell death characterized by leakage of intracellular content to the extracellular space. ASC specks are released among cytosolic content, and accumulate in tissues of patients with chronic inflammation. However, if extracellular ASC specks contribute to disease, or are merely inert remnants of cell death remains unknown. Here, we show that camelid-derived nanobodies against ASC (VHH ASC ) target and disassemble post-pyroptotic inflammasomes, neutralizing their prionoid, and inflammatory functions. Notably, pyroptosis-driven membrane perforation and exposure of ASC specks to the extracellular environment allowed VHH ASC to target inflammasomes while preserving pre-pyroptotic IL-1b release, essential to host defense. Systemically administrated mouse-specific VHH ASC attenuated inflammation and clinical gout, and antigen-induced arthritis disease. Hence, VHH ASC neutralized post-pyroptotic inflammasomes revealing a previously unappreciated role for these complexes in disease. VHH ASC are the first biologicals that disassemble pre-formed inflammasomes while preserving their functions in host defense.