Mathematical modeling supports the presence of neutrophil depriming in vivo.

Abstract Following migration into the intestinal mucosa in inflammatory bowel disease (IBD), neutrophils enter the intestinal lumen and are excreted. This provides a basis for quantification of disease activity by measuring excreted label following injection of In-111-labeled neutrophils. In severe pan-colitis, 50% of the injected In-111 is typically recovered in the feces, indicating that 50% of neutrophil turnover is via fecal excretion. Neutrophils have an intravascular lifespan of ~10 h and a distribution volume of ~10 L, so total body neutrophil turnover is 10.N/10 cells/h, where N is the peripheral blood neutrophil count (cells/L). Neutrophil loss via the colon in a patient with 50% fecal In-111 loss is therefore N/120 cells/min. Pan-colonic mucosal-blood flow in pan-colitis is 200 mL/min, which would deliver N/5 neutrophils to the colon per min. Therefore, 5/120, or 4%, of incoming neutrophils undergo migration into inflamed bowel. If the 96% of nonmigrating cells exit in a primed state, then at steady state >90% of circulating neutrophils would be primed if no depriming took place. As the highest level of priming seen in IBD is ~40%, this indicates that depriming within the circulation must take place. Using the above values in the steady state equation relating priming rate to depriming rate plus primed-cell destruction rate gives a mean depriming time of 35 min. We conclude that a very small proportion of neutrophils entering a site of inflammation migrate and that in vivo depriming must take place to limit the numbers of primed neutrophils in the circulation.This study was supported by the Wellcome Trust and the UK‐US Fulbright Commission; CS holds a Wellcome Trust Postdoctoral Clinical Research Fellowship and a Fulbright Scholar award. The work in the Chilvers lab is funded by the Wellcome Trust, MRC, Asthma‐UK, BBSRC, Gates Foundation and NIHR Cambridge BRC.This is the final published version, also available from http://physreports.physiology.org/content/2/3/e00241.long

Alveolar Macrophages Isolated Directly From Human Cytomegalovirus (HCMV)-Seropositive Individuals Are Sites of HCMV Reactivation In Vivo.

Human cytomegalovirus (HCMV) causes significant morbidity in the immunocompromised host. Following primary infection, the virus establishes latent infection in progenitor cells of the myeloid lineage. These cells exhibit limited viral gene transcription and no evidence of de novo virion production. It is well recognized that differentiation of latently infected myeloid progenitor cells to dendritic or macrophage-like cells permits viral reactivation in vitro. This has been used to support the concept that viral reactivation in HCMV carriers routinely occurs from such terminally differentiated myeloid cells in vivo. However, to date this has not been shown for in vivo-differentiated macrophages. This study is the first to demonstrate that alveolar macrophages from HCMV carriers express immediate early lytic genes and produce infectious virus. This supports the view, until now based on in vitro data, that terminally differentiated myeloid cells in vivo are sites of HCMV reactivation and potential centers of viral dissemination in latently infected individuals with no evidence of virus disease or dissemination.This work was supported by the UK Medical Research Council (grant 0701279 to J. S.) and the National Institute for Health Research UK Biomedical Research Centre (to J. S. and E. R. C.).This is the final published version. It first appeared at http://jid.oxfordjournals.org/content/211/12/1936

Phenotypically distinct neutrophils patrol uninfected human and mouse lymph nodes.

Neutrophils play a key role in innate immunity. As the dominant circulating phagocyte, they are rapidly recruited from the bloodstream to sites of infection or injury to internalize and destroy microbes. More recently, neutrophils have been identified in uninfected organs, challenging the classical view of their function. Here we show that neutrophils were present in lymph nodes (LNs) in homeostasis. Using flow cytometry and confocal imaging, we identified neutrophils within LNs in naive, unchallenged mice, including LNs draining the skin, lungs, and gastrointestinal tract. Neutrophils were enriched within specific anatomical regions, in the interfollicular zone, a site of T cell activation. Intravital two-photon microscopy demonstrated that LN neutrophils were motile, trafficked into LNs from both blood and tissues via high endothelial venules and afferent lymphatics, respectively, and formed interactions with dendritic cells in LNs. Murine and human LN neutrophils had a distinct phenotype compared with circulating neutrophils, with higher major histocompatibility complex II (MHCII) expression, suggesting a potential role in CD4 T cell activation. Upon ex vivo stimulation with IgG immune complex (IC), neutrophils up-regulated expression of MHCII and costimulatory molecules and increased T cell activation. In vivo, neutrophils were capable of delivering circulating IC to LNs, suggesting a broader functional remit. Overall, our data challenge the perception that neutrophil patrol is limited to the circulation in homeostasis, adding LNs to their routine surveillance territory.L.S.C.L. was funded by Wellcome Trust (104384/Z/14/Z). M.R.C. is supported by National Institute of Health Research (NIHR) Cambridge Biomedical Research Centre, Chan-Zuckerburg Initiative Human Cell Atlas Technology Development Grant, Medical Research Council New Investigator Research Grant (MR/N024907/1), Arthritis Research UK Cure Challenge Research Grant (21777), and NIHR Research Professorship (RP-2017-08- ST2-002)

Pulmonary retention of primed neutrophils: a novel protective host response, which is impaired in the acute respiratory distress syndrome.

RATIONALE: Acute respiratory distress syndrome (ARDS) affects over 200000 people annually in the USA. Despite causing severe, and often refractory, hypoxaemia, the high mortality and long-term morbidity of ARDS results mainly from extra-pulmonary organ failure; however the mechanism for this organ crosstalk has not been determined. METHODS: Using autologous radiolabelled neutrophils we investigated the pulmonary transit of primed and unprimed neutrophils in humans. Flow cytometry of whole blood samples was used to assess transpulmonary neutrophil priming gradients in patients with ARDS, sepsis and perioperative controls. MAIN RESULTS: Unprimed neutrophils passed through the lungs with a transit time of 14.2 s, only 2.3 s slower than erythrocytes, and with <5% first-pass retention. Over 97% of neutrophils primed ex vivo with granulocyte macrophage colony-stimulating factor were retained on first pass, with 48% still remaining in the lungs at 40 min. Neutrophils exposed to platelet-activating factor were initially retained but subsequently released such that only 14% remained in the lungs at 40 min. Significant transpulmonary gradients of neutrophil CD62L cell surface expression were observed in ARDS compared with perioperative controls and patients with sepsis. CONCLUSIONS: We demonstrated minimal delay and retention of unprimed neutrophils transiting the healthy human pulmonary vasculature, but marked retention of primed neutrophils; these latter cells then 'deprime' and are re-released into the systemic circulation. Further, we show that this physiological depriming mechanism may fail in patients with ARDS, resulting in increased numbers of primed neutrophils within the systemic circulation. This identifies a potential mechanism for the remote organ damage observed in patients with ARDS.This work was supported by the Wellcome Trust, MRC (UK), Papworth Hospital R&D, Intensive Care Society and NIHR Cambridge Biomedical Research Centre.This is the final published version, also available from http://thorax.bmj.com/content/early/2014/04/04/thoraxjnl-2013-204742.full

Intravital Imaging of Adoptive T-Cell Morphology, Mobility and Trafficking Following Immune Checkpoint Inhibition in a Mouse Melanoma Model

Efficient T-cell targeting, infiltration and activation within tumors is crucial for successful adoptive T-cell therapy. Intravital microscopy is a powerful tool for the visualization of T-cell behavior within tumors, as well as spatial and temporal heterogeneity in response to immunotherapy. Here we describe an experimental approach for intravital imaging of adoptive T-cell morphology, mobility and trafficking in a skin-flap tumor model, following immune modulation with immune checkpoint inhibitors (ICIs) targeting PD-L1 and CTLA-4. A syngeneic model of ovalbumin and mCherry-expressing amelanotic mouse melanoma was used in conjunction with adoptively transferred OT-1+ cytotoxic T-cells expressing GFP to image antigen-specific live T-cell behavior within the tumor microenvironment. Dynamic image analysis of T-cell motility showed distinct CD8+ T-cell migration patterns and morpho-dynamics within different tumor compartments in response to ICIs: this approach was used to cluster T-cell behavior into four groups based on velocity and meandering index. The results showed that most T-cells within the tumor periphery demonstrated Lévy-like trajectories, consistent with tumor cell searching strategies. T-cells adjacent to tumor cells had reduced velocity and appeared to probe the local environment, consistent with cell-cell interactions. An increased number of T-cells were detected following treatment, traveling at lower mean velocities than controls, and demonstrating reduced displacement consistent with target engagement. Histogram-based analysis of immunofluorescent images from harvested tumors showed that in the ICI-treated mice there was a higher density of CD31+ vessels compared to untreated controls and a greater infiltration of T-cells towards the tumor core, consistent with increased cellular trafficking post-treatment

Cardiovascular adaptation to hypoxia and the role of peripheral resistance.

Systemic vascular pressure in vertebrates is regulated by a range of factors: one key element of control is peripheral resistance in tissue capillary beds. Many aspects of the relationship between central control of vascular flow and peripheral resistance are unclear. An important example of this is the relationship between hypoxic response in individual tissues, and the effect that response has on systemic cardiovascular adaptation to oxygen deprivation. We show here how hypoxic response via the HIF transcription factors in one large vascular bed, that underlying the skin, influences cardiovascular response to hypoxia in mice. We show that the response of the skin to hypoxia feeds back on a wide range of cardiovascular parameters, including heart rate, arterial pressures, and body temperature. These data represent the first demonstration of a dynamic role for oxygen sensing in a peripheral tissue directly modifying cardiovascular response to the challenge of hypoxia

Incidence and recognition of acute respiratory distress syndrome in a UK intensive care unit.

The reported incidence of ARDS is highly variable (2.5%-19% of intensive care unit (ICU) patients) and varies depending on study patient population used. We undertook a 6-month, prospective study to determine the incidence and outcome of ARDS in a UK adult University Hospital ICU. 344 patients were admitted during the study period, of these 43 (12.5%) were determined to have ARDS. Patients with ARDS had increased mortality at 28 days and 2 years post-diagnosis, and there was under-recognition of ARDS in both medical records and death certificattion. Our findings have implications for critical care resource planning.This is the final version of the article. It first appeared from BMJ Thorax via ://dx.doi.org/10.1136/thoraxjnl-2016-20840

Viscoelastic properties of differentiating blood cells are fate- and function-dependent.

Although cellular mechanical properties are known to alter during stem cell differentiation, understanding of the functional relevance of such alterations is incomplete. Here, we show that during the course of differentiation of human myeloid precursor cells into three different lineages, the cells alter their viscoelastic properties, measured using an optical stretcher, to suit their ultimate fate and function. Myeloid cells circulating in blood have to be advected through constrictions in blood vessels, engendering the need for compliance at short time-scales (minutes), compared to undifferentiated cells. These findings suggest that reduction in steady-state viscosity is a physiological adaptation for enhanced migration through tissues. Our results indicate that the material properties of cells define their function, can be used as a cell differentiation marker and could serve as target for novel therapies

Clinical application of autologous technetium-99m-labelled eosinophils to detect focal eosinophilic inflammation in the lung.

This is the final version of the article. It first appeared from the BMJ Group via http://dx.doi.org/10.1136/thoraxjnl-2015-207156The detection of focal eosinophilic inflammation by non-invasive means may aid the diagnosis and follow-up of a variety of pulmonary pathologies. All current methods of detection involve invasive sampling, which may be contraindicated or too high-risk to be performed safely. The use of injected autologous technetium-99m (Tc-99m)-labelled eosinophils coupled to single-photon emission computed tomography (SPECT) has been demonstrated to localise eosinophilic inflammation in the lungs of a patient with antineutrophil cytoplasmic antibody-positive vasculitis. Here, we report on the utility of this technique to detect active eosinophilic inflammation in a patient with focal lung inflammation where a biopsy was contraindicated.The authors thank all the staff at the Department of Nuclear Medicine at Addenbrooke’s Hospital and the Wellcome Trust Clinical Research Facility, Cambridge; Cambridge Biomedical Research Centre Core Biochemistry Assay Laboratory; and the National Institute for Health Research, through the Comprehensive Clinical Research Network. This work was supported by Asthma-UK [08/11], the Medical Research Council [grant number MR/J00345X/1], the Wellcome Trust [grant number 098351/Z/12/Z], and Cambridge NIHR Biomedical Research Centre. Written informed consent was obtained in accordance with the Declaration of Helsinki. The study was approved by Cambridgeshire Research Ethics Committee (09/H0308/119) and the Administration of Radioactive Substances Advisory Committee of the United Kingdom (83/3130/25000)

Real-time deformability cytometry reveals sequential contraction and expansion during neutrophil priming.

It has become increasingly apparent that the biomechanical properties of neutrophils impact on their trafficking through the circulation and in particularly through the pulmonary capillary bed. The retention of polarized or shape-changed neutrophils in the lungs was recently proposed to contribute to acute respiratory distress syndrome pathogenesis. Accordingly, this study tested the hypothesis that neutrophil priming is coupled to morpho-rheological (MORE) changes capable of altering cell function. We employ real-time deformability cytometry (RT-DC), a recently developed, rapid, and sensitive way to assess the distribution of size, shape, and deformability of thousands of cells within seconds. During RT-DC analysis, neutrophils can be easily identified within anticoagulated "whole blood" due to their unique granularity and size, thus avoiding the need for further isolation techniques, which affect biomechanical cell properties. Hence, RT-DC is uniquely suited to describe the kinetics of MORE cell changes. We reveal that, following activation or priming, neutrophils undergo a short period of cell shrinking and stiffening, followed by a phase of cell expansion and softening. In some contexts, neutrophils ultimately recover their un-primed mechanical phenotype. The mechanism(s) underlying changes in human neutrophil size are shown to be Na+ /H+ antiport-dependent and are predicted to have profound implications for neutrophil movement through the vascular system in health and disease