Although antimicrobial histones have been isolated from multiple metazoan species, their role in host defense has long remained unanswered. We found here that the hemocytes of the oyster Crassostrea gigas release antimicrobial H1-like and H5-like histones in response to tissue damage and infection. These antimicrobial histones were shown to be associated with extracellular DNA networks released by hemocytes, the circulating immune cells of invertebrates, in response to immune challenge. The hemocyte-released DNA was found to surround and entangle vibrios. This defense mechanism is reminiscent of the neutrophil extracellular traps (ETs) recently described in vertebrates. Importantly, oyster ETs were evidenced in vivo in hemocyte-infiltrated interstitial tissues surrounding wounds, whereas they were absent from tissues of unchallenged oysters. Consistently, antimicrobial histones were found to accumulate in oyster tissues following injury or infection with vibrios. Finally, oyster ET formation was highly dependent on the production of reactive oxygen species by hemocytes. This shows that ET formation relies on common cellular and molecular mechanisms from vertebrates to invertebrates. Altogether, our data reveal that ET formation is a defense mechanism triggered by infection and tissue damage, which is shared by relatively distant species suggesting either evolutionary conservation or convergent evolution within Bilateria.

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Oysters are sessile filter feeders that live in close association with abundant and diverse communities of microorganisms that form the oyster microbiota. In such an association, cellular and molecular mechanisms have evolved to maintain oyster homeostasis upon stressful conditions including infection and changing environments. We give here cellular and molecular insights into the Crassostrea gigas antimicrobial defense system with focus on antimicrobial peptides and proteins (AMPs). This review highlights the central role of the hemocytes in the modulation and control of oyster antimicrobial response. As vehicles for AMPs and other antimicrobial effectors, including reactive oxygen species (ROS), and together with epithelia, hemocytes provide the oyster with local defense reactions instead of systemic humoral ones. These reactions are largely based on phagocytosis but also, as recently described, on the extracellular release of antimicrobial histones (ETosis) which is triggered by ROS. Thus, ROS can signal danger and activate cellular responses in the oyster. From the current literature, AMP production/release could serve similar functions. We provide also new lights on the oyster genetic background that underlies a great diversity of AMP sequences but also an extraordinary individual polymorphism of AMP gene expression. We discuss here how this polymorphism could generate new immune functions, new pathogen resistances or support individual adaptation to environmental stresses.

Recent studies revealed that several vibrio species have evolved the capacity to survive inside host cells. However, it is still often ignored if intracellular stages are required for pathogenicity. Virulence of Vibrio tasmaniensis LGP32, a strain pathogenic for Crassostrea gigas oysters, depends on entry into hemocytes, the oyster immune cells. We investigated here the mechanisms of LGP32 intracellular survival and their consequences on the host-pathogen interaction. Entry and survival inside hemocytes were required for LGP32-driven cytolysis of hemocytes, both in vivo and in vitro. LGP32 intracellular stages showed a profound boost in metabolic activity and a major transcription of antioxidant and copper detoxification genes, as revealed by RNA sequencing. LGP32 isogenic mutants showed that resistance to oxidative stress and copper efflux are two main functions required for vibrio intracellular stages and cytotoxicity to hemocytes. Copper efflux was also essential for host colonization and virulence in vivo. Altogether, our results identify copper resistance as a major mechanism to resist killing by phagocytes, induce cytolysis of immune cells and colonize oysters. Selection of such resistance traits could arise from vibrio interactions with copper-rich environmental niches including marine invertebrates, which favour the emergence of pathogenic vibrios resistant to intraphagosomal killing across animal species.

Delivery of cell therapies often requires the ability to hold products in readiness whilst logistical, regulatory and potency considerations are dealt with and recorded. This requires reversibly stopping biological time, a process which is often achieved by cryopreservation. However, cryopreservation itself poses many biological and biophysical challenges to living cells that need to be understood in order to apply the low temperature technologies to their best advantage. This review sets out the history of applied cryopreservation, our current understanding of the various processes involved in storage at cryogenic temperatures, and challenges for robust and reliable uses of cryopreservation within the cell therapy arena.