A basophil-neuronal axis promotes itch.

Itch is an evolutionarily conserved sensation that facilitates expulsion of pathogens and noxious stimuli from the skin. However, in organ failure, cancer, and chronic inflammatory disorders such as atopic dermatitis (AD), itch becomes chronic, intractable, and debilitating. In addition to chronic itch, patients often experience intense acute itch exacerbations. Recent discoveries have unearthed the neuroimmune circuitry of itch, leading to the development of anti-itch treatments. However, mechanisms underlying acute itch exacerbations remain overlooked. Herein, we identify that a large proportion of patients with AD harbor allergen-specific immunoglobulin E (IgE) and exhibit a propensity for acute itch flares. In mice, while allergen-provoked acute itch is mediated by the mast cell-histamine axis in steady state, AD-associated inflammation renders this pathway dispensable. Instead, a previously unrecognized basophil-leukotriene (LT) axis emerges as critical for acute itch flares. By probing fundamental itch mechanisms, our study highlights a basophil-neuronal circuit that may underlie a variety of neuroimmune processes.

Sensory Neurons Co-opt Classical Immune Signaling Pathways to Mediate Chronic Itch.

Mammals have evolved neurophysiologic reflexes, such as coughing and scratching, to expel invading pathogens and noxious environmental stimuli. It is well established that these responses are also associated with chronic inflammatory diseases, including asthma and atopic dermatitis. However, the mechanisms by which inflammatory pathways promote sensations such as itch remain poorly understood. Here, we show that type 2 cytokines directly activate sensory neurons in both mice and humans. Further, we demonstrate that chronic itch is dependent on neuronal IL-4Rα and JAK1 signaling. We also observe that patients with recalcitrant chronic itch that failed other immunosuppressive therapies markedly improve when treated with JAK inhibitors. Thus, signaling mechanisms previously ascribed to the immune system may represent novel therapeutic targets within the nervous system. Collectively, this study reveals an evolutionarily conserved paradigm in which the sensory nervous system employs classical immune signaling pathways to influence mammalian behavior.

Necroptosis triggers spatially restricted neutrophil-mediated vascular damage during lung ischemia reperfusion injury.

Ischemia reperfusion injury represents a common pathological condition that is triggered by the release of endogenous ligands. While neutrophils are known to play a critical role in its pathogenesis, the tissue-specific spatiotemporal regulation of ischemia-reperfusion injury is not understood. Here, using oxidative lipidomics and intravital imaging of transplanted mouse lungs that are subjected to severe ischemia reperfusion injury, we discovered that necroptosis, a nonapoptotic form of cell death, triggers the recruitment of neutrophils. During the initial stages of inflammation, neutrophils traffic predominantly to subpleural vessels, where their aggregation is directed by chemoattractants produced by nonclassical monocytes that are spatially restricted in this vascular compartment. Subsequent neutrophilic disruption of capillaries resulting in vascular leakage is associated with impaired graft function. We found that TLR4 signaling in vascular endothelial cells and downstream NADPH oxidase 4 expression mediate the arrest of neutrophils, a step upstream of their extravasation. Neutrophil extracellular traps formed in injured lungs and their disruption with DNase prevented vascular leakage and ameliorated primary graft dysfunction. Thus, we have uncovered mechanisms that regulate the initial recruitment of neutrophils to injured lungs, which result in selective damage to subpleural pulmonary vessels and primary graft dysfunction. Our findings could lead to the development of new therapeutics that protect lungs from ischemia reperfusion injury.

Post-exposure intranasal IFNα suppresses replication and neuroinvasion of Venezuelan Equine Encephalitis virus within olfactory sensory neurons.

BACKGROUND: Venezuelan Equine Encephalitis virus (VEEV) may enter the central nervous system (CNS) within olfactory sensory neurons (OSN) that originate in the nasal cavity after intranasal exposure. While it is known that VEEV has evolved several mechanisms to inhibit type I interferon (IFN) signaling within infected cells, whether this inhibits virologic control during neuroinvasion along OSN has not been studied. METHODS: We utilized an established murine model of intranasal infection with VEEV and a repository of scRNAseq data from IFN-treated OSN to assess the cellular targets and IFN signaling responses after VEEV exposure. RESULTS: We found that immature OSN, which express higher levels of the VEEV receptor LDLRAD3 than mature OSN, are the first cells infected by VEEV. Despite rapid VEEV neuroinvasion after intranasal exposure, olfactory neuroepithelium (ONE) and olfactory bulb (OB) IFN responses, as assessed by evaluation of expression of interferon signaling genes (ISG), are delayed for up to 48 h during VEEV neuroinvasion, representing a potential therapeutic window. Indeed, a single intranasal dose of recombinant IFNα triggers early ISG expression in both the nasal cavity and OB. When administered at the time of or early after infection, IFNα treatment delayed onset of sequelae associated with encephalitis and extended survival by several days. VEEV replication after IFN treatment was also transiently suppressed in the ONE, which inhibited subsequent invasion into the CNS. CONCLUSIONS: Our results demonstrate a critical and promising first evaluation of intranasal IFNα for the treatment of human encephalitic alphavirus exposures.

Listeria motility increases the efficiency of epithelial invasion during intestinal infection.

Listeria monocytogenes (Lm) is a food-borne pathogen that causes severe bacterial gastroenteritis, with high rates of hospitalization and mortality. Lm is ubiquitous in soil, water and livestock, and can survive and proliferate at low temperatures. Following oral ingestion of contaminated food, Lm crosses the epithelium through intestinal goblet cells in a mechanism mediated by Lm InlA binding host E-cadherin. Importantly, human infections typically occur with Lm growing at or below room temperature, which is flagellated and motile. Even though many important human bacterial pathogens are flagellated, little is known regarding the effect of Lm motility on invasion and immune evasion. Here, we used complementary imaging and computer modeling approaches to test the hypothesis that bacterial motility helps Lm locate and engage target cells permissive for invasion. Imaging explanted mouse and human intestine, we showed that Lm grown at room temperature uses motility to scan the epithelial surface and preferentially attach to target cells. Furthermore, we integrated quantitative parameters from our imaging experiments to construct a versatile "layered" cellular Potts model (L-CPM) that simulates host-pathogen dynamics. Simulated data are consistent with the hypothesis that bacterial motility enhances invasion by allowing bacteria to search the epithelial surface for their preferred invasion targets. Indeed, our model consistently predicts that motile bacteria invade twice as efficiently over the first hour of infection. We also examined how bacterial motility affected interactions with host cellular immunity. In a mouse model of persistent infection, we found that neutrophils migrated to the apical surface of the epithelium 5 hours post infection and interacted with Lm. Yet in contrast to the view that neutrophils "hunt" for bacteria, we found that these interactions were driven by motility of Lm-which moved at least ~50x faster than neutrophils. Furthermore, our L-CPM predicts that motile bacteria maintain their invasion advantage even in the presence of host phagocytes, with the balance between invasion and phagocytosis governed almost entirely by bacterial motility. In conclusion, our simulations provide insight into host pathogen interaction dynamics at the intestinal epithelial barrier early during infection.

Phagocytes produce prostaglandin E2 in response to cytosolic Listeria monocytogenes.

Listeria monocytogenes is an intracellular bacterium that elicits robust CD8+ T-cell responses. Despite the ongoing development of L. monocytogenes-based platforms as cancer vaccines, our understanding of how L. monocytogenes drives robust CD8+ T-cell responses remains incomplete. One overarching hypothesis is that activation of cytosolic innate pathways is critical for immunity, as strains of L. monocytogenes that are unable to access the cytosol fail to elicit robust CD8+ T-cell responses and in fact inhibit optimal T-cell priming. Counterintuitively, however, activation of known cytosolic pathways, such as the inflammasome and type I IFN, lead to impaired immunity. Conversely, production of prostaglandin E2 (PGE2) downstream of cyclooxygenase-2 (COX-2) is essential for optimal L. monocytogenes T-cell priming. Here, we demonstrate that vacuole-constrained L. monocytogenes elicit reduced PGE2 production compared to wild-type strains in macrophages and dendritic cells ex vivo. In vivo, infection with wild-type L. monocytogenes leads to 10-fold increases in PGE2 production early during infection whereas vacuole-constrained strains fail to induce PGE2 over mock-immunized controls. Mice deficient in COX-2 specifically in Lyz2+ or CD11c+ cells produce less PGE2, suggesting these cell subsets contribute to PGE2 levels in vivo, while depletion of phagocytes with clodronate abolishes PGE2 production completely. Taken together, this work demonstrates that optimal PGE2 production by phagocytes depends on L. monocytogenes access to the cytosol, suggesting that one reason cytosolic access is required to prime CD8+ T-cell responses may be to facilitate production of PGE2.

Enterotoxigenic Escherichia coli Degrades the Host MUC2 Mucin Barrier To Facilitate Critical Pathogen-Enterocyte Interactions in Human Small Intestine.

Enterotoxigenic Escherichia coli (ETEC) isolates are genetically diverse pathological variants of E. coli defined by the production of heat-labile (LT) and/or heat-stable (ST) toxins. ETEC strains are estimated to cause hundreds of millions of cases of diarrheal illness annually. However, it is not clear that all strains are equally equipped to cause disease, and asymptomatic colonization with ETEC is common in low- to middle-income regions lacking basic sanitation and clean water where ETEC are ubiquitous. Recent molecular epidemiology studies have revealed a significant association between strains that produce EatA, a secreted autotransporter protein, and the development of symptomatic infection. Here, we demonstrate that LT stimulates production of MUC2 mucin by goblet cells in human small intestine, enhancing the protective barrier between pathogens and enterocytes. In contrast, using explants of human small intestine as well as small intestinal enteroids, we show that EatA counters this host defense by engaging and degrading the MUC2 mucin barrier to promote bacterial access to target enterocytes and ultimately toxin delivery, suggesting that EatA plays a crucial role in the molecular pathogenesis of ETEC. These findings may inform novel approaches to prevention of acute diarrheal illness as well as the sequelae associated with ETEC and other pathogens that rely on EatA and similar proteases for efficient interaction with their human hosts.

CD8α(+) dendritic cells are an obligate cellular entry point for productive infection by Listeria monocytogenes.

CD8α(+) dendritic cells (DCs) prime cytotoxic T lymphocytes during viral infections and produce interleukin-12 in response to pathogens. Although the loss of CD8α(+) DCs in Batf3(-/-) mice increases their susceptibility to several pathogens, we observed that Batf3(-/-) mice exhibited enhanced resistance to the intracellular bacterium Listeria monocytogenes. In wild-type mice, Listeria organisms, initially located in the splenic marginal zone, migrated to the periarteriolar lymphoid sheath (PALS) where they grew exponentially and induced widespread lymphocyte apoptosis. In Batf3(-/-) mice, however, Listeria organisms remain trapped in the marginal zone, failed to traffic into the PALS, and were rapidly cleared by phagocytes. In addition, Batf3(-/-) mice, which lacked the normal population of hepatic CD103(+) peripheral DCs, also showed protection from liver infection. These results suggest that Batf3-dependent CD8α(+) and CD103(+) DCs provide initial cellular entry points within the reticuloendothelial system by which Listeria establishes productive infection.

Genetic Regulation of Fibroblast Activation and Proliferation in Cardiac Fibrosis.

BACKGROUND: Genetic diversity and the heterogeneous nature of cardiac fibroblasts (CFbs) have hindered characterization of the molecular mechanisms that regulate cardiac fibrosis. The Hybrid Mouse Diversity Panel offers a valuable tool to examine genetically diverse cardiac fibroblasts and their role in fibrosis. METHODS: Three strains of mice (C57BL/6J, C3H/HeJ, and KK/HlJ) were selected from the Hybrid Mouse Diversity Panel and treated with either isoproterenol (ISO) or saline by an intraperitoneally implanted osmotic pump. After 21 days, cardiac function and levels of fibrosis were measured by echocardiography and trichrome staining, respectively. Activation and proliferation of CFbs were measured by in vitro and in vivo assays under normal and injury conditions. RNA sequencing was done on isolated CFbs from each strain. Results were analyzed by Ingenuity Pathway Analysis and validated by reverse transcription-qPCR, immunohistochemistry, and ELISA. RESULTS: ISO treatment in C57BL/6J, C3H/HeJ, and KK/HlJ mice resulted in minimal, moderate, and extensive levels of fibrosis, respectively (n=7-8 hearts per condition). Isolated CFbs treated with ISO exhibited strain-specific increases in the levels of activation but showed comparable levels of proliferation. Similar results were found in vivo, with fibroblast activation, and not proliferation, correlating with the differential levels of cardiac fibrosis after ISO treatment. RNA sequencing revealed that CFbs from each strain exhibit unique gene expression changes in response to ISO. We identified Ltbp2 as a commonly upregulated gene after ISO treatment. Expression of LTBP2 was elevated and specifically localized in the fibrotic regions of the myocardium after injury in mice and in human heart failure patients. CONCLUSIONS: This study highlights the importance of genetic variation in cardiac fibrosis by using multiple inbred mouse strains to characterize CFbs and their response to ISO treatment. Our data suggest that, although fibroblast activation is a response that parallels the extent of scar formation, proliferation may not necessarily correlate with levels of fibrosis. In addition, by comparing CFbs from multiple strains, we identified pathways as potential therapeutic targets and LTBP2 as a marker for fibrosis, with relevance to patients with underlying myocardial fibrosis.

Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance.

Neutrophil extracellular traps (NETs) have been shown to worsen acute pulmonary injury including after lung transplantation. The breakdown of NETs by DNAse-1 can help restore lung function, but whether there is an impact on allograft tolerance remains less clear. Using intravital 2-photon microscopy, we analyzed the effects of DNAse-1 on NETs in mouse orthotopic lung allografts damaged by ischemia-reperfusion injury. Although DNAse-1 treatment rapidly degrades intragraft NETs, the consequential release of NET fragments induces prolonged interactions between infiltrating CD4+ T cells and donor-derived antigen presenting cells. DNAse-1 generated NET fragments also promote human alveolar macrophage inflammatory cytokine production and prime dendritic cells for alloantigen-specific CD4+ T cell proliferation through activating toll-like receptor (TLR) - Myeloid Differentiation Primary Response 88 (MyD88) signaling pathways. Furthermore, and in contrast to allograft recipients with a deficiency in NET generation due to a neutrophil-specific ablation of Protein Arginine Deiminase 4 (PAD4), DNAse-1 administration to wild-type recipients promotes the recognition of allo- and self-antigens and prevents immunosuppression-mediated lung allograft acceptance through a MyD88-dependent pathway. Taken together, these data show that the rapid catalytic release of NET fragments promotes innate immune responses that prevent lung transplant tolerance.

Mammalian target of rapamycin controls dendritic cell development downstream of Flt3 ligand signaling.

Dendritic cells (DCs) comprise distinct functional subsets including CD8⁻ and CD8(+) classical DCs (cDCs) and interferon-secreting plasmacytoid DCs (pDCs). The cytokine Flt3 ligand (Flt3L) controls the development of DCs and is particularly important for the pDC and CD8(+) cDC and their CD103(+) tissue counterparts. We report that mammalian target of rapamycin (mTOR) inhibitor rapamycin impaired Flt3L-driven DC development in vitro, with the pDCs and CD8(+)-like cDCs most profoundly affected. Conversely, deletion of the phosphoinositide 3-kinase (PI3K)-mTOR negative regulator Pten facilitated Flt3L-driven DC development in culture. DC-specific Pten targeting in vivo caused the expansion of CD8(+) and CD103(+) cDC numbers, which was reversible by rapamycin. The increased CD8(+) cDC numbers caused by Pten deletion correlated with increased susceptibility to the intracellular pathogen Listeria. Thus, PI3K-mTOR signaling downstream of Flt3L controls DC development, and its restriction by Pten ensures optimal DC pool size and subset composition.

Real-Time Analysis of Calcium Signals during the Early Phase of T Cell Activation Using a Genetically Encoded Calcium Biosensor.

Proper T cell activation is promoted by sustained calcium signaling downstream of the TCR. However, the dynamics of calcium flux after stimulation with an APC in vivo remain to be fully understood. Previous studies focusing on T cell motility suggested that the activation of naive T cells in the lymph node occurs in distinct phases. In phase I, T cells make multiple transient contacts with dendritic cells before entering a phase II, where they exist in stable clusters with dendritic cells. It has been suggested that T cells signal during transient contacts of phase I, but this has never been shown directly. Because time-dependent loss of calcium dyes from cells hampers long-term imaging of cells in vivo after antigenic stimulation, we generated a knock-in mouse expressing a modified form of the Cameleon fluorescence resonance energy transfer reporter for intracellular calcium and examined calcium flux both in vitro and in situ. In vitro, we observed transient, oscillatory, and sustained calcium flux after contact with APC, but these behaviors were not affected by the type of APC or Ag quantity, but were, however, moderately dependent on Ag quality. In vivo, we found that during phase I, T cells exhibit weak calcium fluxes and detectable changes in cell motility. This demonstrates that naive T cells signal during phase I and support the hypothesis that accumulated calcium signals are required to signal the beginning of phase II.

Goblet cells deliver luminal antigen to CD103+ dendritic cells in the small intestine.

The intestinal immune system is exposed to a mixture of foreign antigens from diet, commensal flora and potential pathogens. Understanding how pathogen-specific immunity is elicited while avoiding inappropriate responses to the background of innocuous antigens is essential for understanding and treating intestinal infections and inflammatory diseases. The ingestion of protein antigen can induce oral tolerance, which is mediated in part by a subset of intestinal dendritic cells (DCs) that promote the development of regulatory T cells. The lamina propria (LP) underlies the expansive single-cell absorptive villous epithelium and contains a large population of DCs (CD11c(+) CD11b(+) MHCII(+) cells) comprised of two predominant subsets: CD103(+) CX(3)CR1(-) DCs, which promote IgA production, imprint gut homing on lymphocytes and induce the development of regulatory T cells, and CD103(-) CX(3)CR1(+) DCs (with features of macrophages), which promote tumour necrosis factor-α (TNF-α) production, colitis, and the development of T(H)17 T cells. However, the mechanisms by which different intestinal LP-DC subsets capture luminal antigens in vivo remains largely unexplored. Using a minimally disruptive in vivo imaging approach we show that in the steady state, small intestine goblet cells (GCs) function as passages delivering low molecular weight soluble antigens from the intestinal lumen to underlying CD103(+) LP-DCs. The preferential delivery of antigens to DCs with tolerogenic properties implies a key role for this GC function in intestinal immune homeostasis.

CCR6 promotes steady-state mononuclear phagocyte association with the intestinal epithelium, imprinting and immune surveillance.

The intestinal lamina propria (LP) contains antigen-presenting cells with features of dendritic cells and macrophages, collectively referred to as mononuclear phagocytes (MNPs). Association of MNPs with the epithelium is thought to play an important role in multiple facets of intestinal immunity including imprinting MNPs with the ability to induce IgA production, inducing the expression of gut homing molecules on T cells, facilitating the capture of luminal antigens and microbes, and subsequent immune responses in the mesenteric lymph node (MLN). However, the factors promoting this process in the steady state are largely unknown, and in vivo models to test and confirm the importance of LP-MNP association with the epithelium for these outcomes are unexplored. Evaluation of epithelial expression of chemoattractants in mice where MNP-epithelial associations were impaired suggested CCL20 as a candidate promoting epithelial association. Expression of CCR6, the only known receptor for CCL20, was required for MNPs to associate with the epithelium. LP-MNPs from CCR6-/- mice did not display defects in acquiring antigen and stimulating T-cell responses in ex vivo assays or in responses to antigen administered systemically. However, LP-MNPs from CCR6-deficient mice were impaired at acquiring luminal and epithelial antigens, inducing IgA production in B cells, inducing immune responses in the MLN, and capturing and trafficking luminal commensal bacteria to the MLN. These findings identify a crucial role for CCR6 in promoting LP-MNPs to associate with the intestinal epithelium in the steady state to perform multiple functions promoting gut immune homeostasis.

Age-Dependent Cell Trafficking Defects in Draining Lymph Nodes Impair Adaptive Immunity and Control of West Nile Virus Infection.

Impaired immune responses in the elderly lead to reduced vaccine efficacy and increased susceptibility to viral infections. Although several groups have documented age-dependent defects in adaptive immune priming, the deficits that occur prior to antigen encounter remain largely unexplored. Herein, we identify novel mechanisms for compromised adaptive immunity that occurs with aging in the context of infection with West Nile virus (WNV), an encephalitic flavivirus that preferentially causes disease in the elderly. An impaired IgM and IgG response and enhanced vulnerability to WNV infection during aging was linked to delayed germinal center formation in the draining lymph node (DLN). Adoptive transfer studies and two-photon intravital microscopy revealed a decreased trafficking capacity of donor naïve CD4+ T cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within DLN prior to antigen encounter. Furthermore, leukocyte accumulation in the DLN within the first few days of WNV infection or antigen-adjuvant administration was diminished more generally in old mice and associated with a second aging-related defect in local cytokine and chemokine production. Thus, age-dependent cell-intrinsic and environmental defects in the DLN result in delayed immune cell recruitment and antigen recognition. These deficits compromise priming of early adaptive immune responses and likely contribute to the susceptibility of old animals to acute WNV infection.

Concerted activity of IgG1 antibodies and IL-4/IL-25-dependent effector cells trap helminth larvae in the tissues following vaccination with defined secreted antigens, providing sterile immunity to challenge infection.

Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.

Bacterial entry to the splenic white pulp initiates antigen presentation to CD8+ T cells.

The spleen plays an important role in host-protective responses to bacteria. However, the cellular dynamics that lead to pathogen-specific immunity remain poorly understood. Here we examined Listeria monocytogenes (Lm) infection in the mouse spleen via in situ fluorescence microscopy. We found that the redistribution of Lm from the marginal zone (MZ) to the periarteriolar lymphoid sheath (PALS) was inhibited by pertussis toxin and required the presence of CD11c(+) cells. As early as 9 hr after infection, we detected infected dendritic cells in the peripheral regions of the PALS and clustering of Lm-specific T cells by two-photon microscopy. Pertussis toxin inhibited both Lm entry into the PALS and antigen presentation to CD8(+) T cells. Our study suggests that splenic dendritic cells rapidly deliver intracellular bacteria to the T cell areas of the white pulp to initiate CD8(+) T cell responses.

DAP12 expression in lung macrophages mediates ischemia/reperfusion injury by promoting neutrophil extravasation.

Neutrophils are critical mediators of innate immune responses and contribute to tissue injury. However, immune pathways that regulate neutrophil recruitment to injured tissues during noninfectious inflammation remain poorly understood. DAP12 is a cell membrane-associated protein that is expressed in myeloid cells and can either augment or dampen innate inflammatory responses during infections. To elucidate the role of DAP12 in pulmonary ischemia/reperfusion injury (IRI), we took advantage of a clinically relevant mouse model of transplant-mediated lung IRI. This technique allowed us to dissect the importance of DAP12 in tissue-resident cells and those that infiltrate injured tissue from the periphery during noninfectious inflammation. Macrophages in both mouse and human lungs that have been subjected to cold ischemic storage express DAP12. We found that donor, but not recipient, deficiency in DAP12 protected against pulmonary IRI. Analysis of the immune response showed that DAP12 promotes the survival of tissue-resident alveolar macrophages and contributes to local production of neutrophil chemoattractants. Intravital imaging demonstrated a transendothelial migration defect into DAP12-deficient lungs, which can be rescued by local administration of the neutrophil chemokine CXCL2. We have uncovered a previously unrecognized role for DAP12 expression in tissue-resident alveolar macrophages in mediating acute noninfectious tissue injury through regulation of neutrophil trafficking.

Dynamic imaging of the immune system: progress, pitfalls and promise.

Both innate and adaptive immunity are dependent on the migratory capacity of myeloid and lymphoid cells. Effector cells of the innate immune system rapidly enter infected tissues, whereas sentinel dendritic cells in these sites mobilize and transit to lymph nodes. In these and other secondary lymphoid tissues, interactions among various cell types promote adaptive humoral and cell-mediated immune responses. Recent advances in light microscopy have allowed direct visualization of these events in living animals and tissue explants, which allows a new appreciation of the dynamics of immune-cell behaviour. In this article, we review the basic techniques and the tools used for in situ imaging, as well as the limitations and potential artefacts of these methods.

Quantifying progression and regression of thrombotic risk in experimental atherosclerosis.

Currently, there are no generally applicable noninvasive methods for defining the relationship between atherosclerotic vascular damage and risk of focal thrombosis. Herein, we demonstrate methods to delineate the progression and regression of vascular damage in response to an atherogenic diet by quantifying the in vivo accumulation of semipermeable 200-300 nm perfluorocarbon core nanoparticles (PFC-NP) in ApoE null mouse plaques with [(19)F] magnetic resonance spectroscopy (MRS). Permeability to PFC-NP remained minimal until 12 weeks on diet, then increased rapidly following 12 weeks, but regressed to baseline within 8 weeks after diet normalization. Markedly accelerated clotting (53.3% decrease in clotting time) was observed in carotid artery preparations of fat-fed mice subjected to photochemical injury as defined by the time to flow cessation. For all mice on and off diet, an inverse linear relationship was observed between the permeability to PFC-NP and accelerated thrombosis (P = 0.02). Translational feasibility for quantifying plaque permeability and vascular damage in vivo was demonstrated with clinical 3 T MRI of PFC-NP accumulating in plaques of atherosclerotic rabbits. These observations suggest that excessive permeability to PFC-NP may indicate prothrombotic risk in damaged atherosclerotic vasculature, which resolves within weeks after dietary therapy.