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.

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

Severe thrombocytopenia can be a determinant factor in the morbidity of Plasmodium vivax, the most widespread human malaria parasite. Although immune mechanisms may drive P. vivax-induced severe thrombocytopenia (PvST), the current data on the cytokine landscape in PvST is scarce and often conflicting. Here, we hypothesized that the analysis of the bidirectional circuit of inflammatory mediators and their regulatory miRNAs would lead to a better understanding of the mechanisms underlying PvST. For that, we combined Luminex proteomics, NanoString miRNA quantification, and machine learning to evaluate an extensive array of plasma mediators in uncomplicated P. vivax patients with different degrees of thrombocytopenia. Unsupervised clustering analysis identified a set of PvST-linked inflammatory (CXCL10, CCL4, and IL-18) and regulatory (IL-10, IL-1Ra, HGF) mediators. Among the mediators associated with PvST, IL-6 and IL-8 were critical to discriminate P. vivax subgroups, while CCL2 and IFN-γ from healthy controls. Supervised machine learning spotlighted IL-10 in P. vivax-mediated thrombocytopenia and provided evidence for a potential signaling route involving IL-8 and HGF. Finally, we identified a set of miRNAs capable of modulating these signaling pathways. In conclusion, the results place IL-10 and IL-8/HGF in the center of PvST and propose investigating these signaling pathways across the spectrum of malaria infections.

Classical CD14+ monocytes are the predominant monocyte population in human blood. They are primarily engaged in host defense programs and secrete pro-inflammatory cytokines that orchestrate immune responses. While aberrant monocyte activity elicits cytokine storms, dysfunctional monocytes are associated with immunoparalysis, an equally life-threatening state of immunosuppression observed in severe sepsis, trauma, or respiratory viral infections. Hence, unraveling the mechanisms controlling monocyte functions is paramount in diverse clinical settings. Here, we reveal a critical dependency on platelets for the pro-inflammatory cytokine responses of human monocytes. We found that platelet removal from freshly isolated primary human monocytes causes monocyte immunoparalysis, characterized by transcriptional shut down of pro-inflammatory genes, and impaired cytokine secretion upon Toll-like and NOD-like receptor activation. Notably, anergic platelet-depleted monocytes can be reactivated upon their replenishment with autologous platelets. Moreover, monocytes from patients with immune thrombocytopenia display naturally impaired cytokine responses, which were also reversed by platelet supplementation. Mechanistically, we show that the trans-cellular propagation of platelet p38 MAPK signaling through platelet vesicles licences the full cytokine responses of human monocytes. Our findings delineate platelets as paramount regulators of monocyte innate immune functions.

Background: Severe thrombocytopenia can be a determinant factor in the morbidity of Plasmodium vivax (Pv), the most widespread human malaria. Although immune mechanisms may drive Pv-induced severe thrombocytopenia (PvST), the current data on the cytokine landscape in PvST is scarce, and often conflicting. The analysis of the bidirectional circuit of inflammatory mediators and miRNAs would lead to a better understanding of the mechanisms underlying PvST. Methods: We combined Luminex proteomics, NanoString miRNA quantification, and machine learning, to evaluate an extensive array of plasma mediators in uncomplicated Pv patients, whose blood platelet counts varied from reference values to PvST. Results: Unsupervised clustering analysis identified PvST-linked signatures comprised of both inflammatory (CXCL10, CCL4, and IL-18) and regulatory (IL-10, IL-1Ra, HGF) mediators. As part of PvST signatures, IL-6 and IL-8 were critical to discriminate Pv subgroups, while CCL2 and IFN-γ from healthy controls. Supervised machine learning spotlighted IL-10 in Pv-mediated thrombocytopenia, and provided evidence for a potential signaling route involving IL-8 and HGF. Finally, we identified a set of miRNAs capable of modulating these signaling pathways. Conclusions: The results place IL-10 and IL-8/HGF in the center of PvST and propose investigating these signaling pathways across the spectrum of malaria infections.