Craig N. Jenne

Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo

Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.Neutrophil extracellular traps (NETs) are released, as neutrophils die in vitro, in a process requiring hours, leaving a temporal gap for invasive microbes to exploit. Functional neutrophils undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live PMN in vivo rapidly releasing NETs, which prevented bacterial dissemination. NETosis occurred during crawling thereby casting large areas of NETs. NET-releasing PMN developed diffuse decondensed nuclei ultimately becoming devoid of DNA. Cells with abnormal nuclei displayed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A combined requirement of Tlr2 and complement mediated opsonization tightly regulated NET release. Additionally live human PMN developed decondensed nuclei and formed NETS in vivo and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection, non-cell death NETosis occurs in vivo during Gram-positive infection in mice and humans.

Intravascular Neutrophil Extracellular Traps Capture Bacteria from the Bloodstream during Sepsis

During the systemic inflammatory response of severe sepsis, neutrophils accumulate in the liver microcirculation, but their functional significance is largely unknown. We show that neutrophils migrate to liver sinusoids during endotoxemia and sepsis where they exert protective effects by releasing neutrophil extracellular traps (NETs), which are DNA-based structures that capture and eliminate microbes. NETs released into the vasculature ensnare bacteria from the bloodstream and prevent dissemination. NET production requires platelet-neutrophil interactions and can be inhibited by platelet depletion or disruption of integrin-mediated platelet-neutrophil binding. During sepsis, NET release increases bacterial trapping by 4-fold (beyond the basal level provided by resident intravascular macrophages). Blocking NET formation reduces the capture of circulating bacteria during sepsis, resulting in increased dissemination to distant organs. Thus, NETs ensnare circulating bacteria and provide intravascular immunity that protects against bacterial dissemination during septic infections.

Immune surveillance by the liver

Receiving both portal vein blood and arterial blood, the liver is an important and critical component in the defense against blood-borne infection. To accomplish this role, the liver contains numerous innate and adaptive immune cells that specialize in detection and capture of pathogens from the blood. Further, these immune cells participate in coordinated immune responses leading to pathogen clearance, leukocyte recruitment and antigen presentation to lymphocytes within the vasculature. Finally, this role in host defense must be tightly regulated to ensure that inappropriate immune responses are not raised against nonpathogenic exogenous blood-borne molecules, such as those derived from food. It is this balance between activation and tolerance that characterizes the liver as a frontline immunological organ.

Functional Innervation of Hepatic iNKT Cells Is Immunosuppressive Following Stroke

Neurotransmitters relay immunosuppressive signals to natural killer T cells after stroke. Systemic immunosuppression has been associated with stroke for many years, but the underlying mechanisms are poorly understood. In this study, we demonstrated that stroke induced profound behavioral changes in hepatic invariant NKT (iNKT) cells in mice. Unexpectedly, these effects were mediated by a noradrenergic neurotransmitter rather than a CD1d ligand or other well-characterized danger signals. Blockade of this innervation was protective in wild-type mice after stroke but had no effect in mice deficient in iNKT cells. Selective immunomodulation of iNKT cells with a specific activator (α-galactosylceramide) promoted proinflammatory cytokine production and prevented infections after stroke. Our results therefore identify a molecular mechanism that leads to immunosuppression after stroke and suggest an attractive potential therapeutic alternative to antibiotics, namely, immunomodulation of iNKT cells to prevent stroke-associated infections.

T-bet–dependent S1P5 expression in NK cells promotes egress from lymph nodes and bone marrow

During a screen for ethylnitrosourea-induced mutations in mice affecting blood natural killer (NK) cells, we identified a strain, designated Duane, in which NK cells were reduced in blood and spleen but increased in lymph nodes (LNs) and bone marrow (BM). The accumulation of NK cells in LNs reflected a decreased ability to exit into lymph. This strain carries a point mutation within Tbx21 (T-bet), which generates a defective protein. Duane NK cells have a 30-fold deficiency in sphingosine-1-phosphate receptor 5 (S1P5) transcript levels, and S1P5-deficient mice exhibit an egress defect similar to Duane. Chromatin immunoprecipitation confirms binding of T-bet to the S1pr5 locus. S1P-deficient mice exhibit a more severe NK cell egress block, and the FTY720-sensitive S1P1 also plays a role in NK cell egress from LNs. S1P5 is not inhibited by CD69, a property that may facilitate trafficking of activated NK cells to effector sites. Finally, the accumulation of NK cells within BM of S1P-deficient mice was associated with reduced numbers in BM sinusoids, suggesting a role for S1P in BM egress. In summary, these findings identify S1P5 as a T-bet–induced gene that is required for NK cell egress from LNs and BM.

Neutrophils Recruited to Sites of Infection Protect from Virus Challenge by Releasing Neutrophil Extracellular Traps

Neutrophils mediate bacterial clearance through various mechanisms, including the release of mesh-like DNA structures or neutrophil extracellular traps (NETs) that capture bacteria. Although neutrophils are also recruited to sites of viral infection, their role in antiviral innate immunity is less clear. We show that systemic administration of virus analogs or poxvirus infection induces neutrophil recruitment to the liver microvasculature and the release of NETs that protect host cells from virus infection. After systemic intravenous poxvirus challenge, mice exhibit thrombocytopenia and the recruitment of both neutrophils and platelets to the liver vasculature. Circulating platelets interact with, roll along, and adhere to the surface of adherent neutrophils, forming large, dynamic aggregates. These interactions facilitate the release of NETs within the liver vasculature that are able to protect host cells from poxvirus infection. These findings highlight the role of NETs and early tissue-wide responses in preventing viral infection.

The actin regulator coronin 1A is mutant in a thymic egress–deficient mouse strain and in a patient with severe combined immunodeficiency

Mice carrying the recessive locus for peripheral T cell deficiency (Ptcd) have a block in thymic egress, but the mechanism responsible is undefined. Here we found that Ptcd T cells had an intrinsic migration defect, impaired lymphoid tissue trafficking and irregularly shaped protrusions. Characterization of the Ptcd locus showed a point substitution of lysine for glutamic acid at position 26 in the actin regulator coronin 1A that enhanced its inhibition of the actin regulator Arp2/3 and resulted in its mislocalization from the leading edge of migrating T cells. The discovery of another coronin 1A mutant during an N-ethyl-N-nitrosourea-mutagenesis screen for T cell–lymphopenic mice prompted us to evaluate a T cell–deficient, B cell–sufficient and natural killer cell–sufficient patient with severe combined immunodeficiency, whom we found had mutations in both CORO1A alleles. Our findings establish a function for coronin 1A in T cell egress, identify a surface of coronin involved in Arp2/3 regulation and demonstrate that actin regulation is a biological process defective in human and mouse severe combined immunodeficiency.

A dynamic spectrum of monocytes arising from the in situ reprogramming of CCR2+ monocytes at a site of sterile injury

In response to sterile liver injury, CCR2hiCX3CR1low inflammatory monocytes infiltrate the liver and form a ringlike structure around the injury site. The cells then transition into CCR2lowCX3CR1hi alternative monocytes that enter the injury site; this phenotypic transition was required for optimal repair.

Platelets: bridging hemostasis, inflammation, and immunity

Although the function of platelets in the maintenance of hemostasis has been studied in great detail, more recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Platelets by virtue of their large numbers and their ability to rapidly release a broad spectrum of immunomodulatory cytokines, chemokines, and other mediators act as circulating sentinels. Upon detection of a pathogen, platelets quickly activate and begin to drive the ensuing inflammatory response. Platelets have the ability to directly modulate the activity of neutrophils (phagocytosis, oxidative burst), endothelium (adhesion molecule and chemokine expression), and lymphocytes. Due to their diverse array of adhesion molecules and preformed chemokines, platelets are able to adhere to leukocytes and facilitate their recruitment to sites of tissue damage or infection. Furthermore, platelets directly participate in the capture and sequestration of pathogens within the vasculature. Platelet–neutrophil interactions are known to induce the release of neutrophil extracellular traps (NETs) in response to either bacterial or viral infection, and platelets have been shown to internalize pathogens, sequestering them in engulfment vacuoles. Finally, emerging data indicate that platelets also participate in the host immune response by directly killing infected cells. This review will highlight the central role platelets play in the initiation and modulation of the host inflammatory and immune responses.

Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature.

Neutrophil extracellular traps (NETs) composed of DNA decorated with histones and proteases trap and kill bacteria but also injure host tissue. Here we show that during a bloodstream infection with methicillin-resistant Staphylococcus aureus, the majority of bacteria are sequestered immediately by hepatic Kupffer cells, resulting in transient increases in liver enzymes, focal ischaemic areas and a robust neutrophil infiltration into the liver. The neutrophils release NETs into the liver vasculature, which remain anchored to the vascular wall via von Willebrand factor and reveal significant neutrophil elastase (NE) proteolytic activity. Importantly, DNase although very effective at DNA removal, and somewhat effective at inhibiting NE proteolytic activity, fails to remove the majority of histones from the vessel wall and only partly reduces injury. By contrast, inhibition of NET production as modelled by PAD4-deficiency, or prevention of NET formation and proteolytic activity as modelled in NE−/− mice prevent collateral host tissue damage.