Jurgen Herre

Allergens as Immunomodulatory Proteins: The Cat Dander Protein Fel d 1 Enhances TLR Activation by Lipid Ligands

Allergic responses can be triggered by structurally diverse allergens. Most allergens are proteins, yet extensive research has not revealed how they initiate the allergic response and why the myriad of other inhaled proteins do not. Among these allergens, the cat secretoglobulin protein Fel d 1 is a major allergen and is responsible for severe allergic responses. In this study, we show that similar to the mite dust allergen Der p 2, Fel d 1 substantially enhances signaling through the innate receptors TLR4 and TLR2. In contrast to Der p 2, however, Fel d 1 does not act by mimicking the TLR4 coreceptor MD2 and is not able to bind stably to the TLR4/MD2 complex in vitro. Fel d 1 does, however, bind to the TLR4 agonist LPS, suggesting that a lipid transfer mechanism may be involved in the Fel d 1 enhancement of TLR signaling. We also show that the dog allergen Can f 6, a member of a distinct class of lipocalin allergens, has very similar properties to Fel d 1. We propose that Fel d 1 and Can f 6 belong to a group of allergen immunomodulatory proteins that enhance innate immune signaling and promote airway hypersensitivity reactions in diseases such as asthma.

Phagocytosis Dynamics Depends on Target Shape

A complete understanding of phagocytosis requires insight into both its biochemical and physical aspects. One of the ways to explore the physical mechanism of phagocytosis is to probe whether and how the target properties (e.g., size, shape, surface states, stiffness, etc.) affect their uptake. Here we report an imaging-based method to explore phagocytosis kinetics, which is compatible with real-time imaging and can be used to validate existing reports using fixed and stained cells. We measure single-event engulfment time from a large number of phagocytosis events to compare how size and shape of targets determine their engulfment. The data shows an increase in the average engulfment time for increased target size, for spherical particles. The uptake time data on nonspherical particles confirms that target shape plays a more dominant role than target size for phagocytosis: Ellipsoids with an eccentricity of 0.954 and much smaller surface areas than spheres were taken up five times more slowly than spherical targets.

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.

Genome-wide transcription profiling in neutrophils in acute respiratory distress syndrome

BACKGROUND Acute respiratory distress syndrome (ARDS) is characterised by diffuse neutrophil-mediated alveolar inflammation. Recently, we demonstrated that blood polymorphonuclear leucocytes (PMNs) in ARDS are basally activated, and exhibit aberrant oxidative burst and survival responses. The molecular mechanisms governing ARDS PMN function and longevity are incompletely understood. We aimed to use genome-wide transcriptional profiling of ARDS blood PMNs to explore underlying disease mechanisms and identify therapeutic targets aimed at manipulating PMN function and longevity. METHODS GeneChip Affymetrix oligonucleotide arrays were used to assess global transcriptional profiles in highly pure PMNs from ventilated patients fulfilling the Berlin ARDS definition (n=10), in freshly isolated PMNs from age-matched and sex-matched healthy volunteers (n=10), and in healthy volunteer PMNs exposed in vitro to recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (1 ng/mL for 6 h). Ingenuity Pathway Analysis software was used to map probes identified as important onto specific pathways. FINDINGS Transcriptomic analysis showed that 1319 genes were altered in ARDS PMNs relative to healthy volunteer PMNs. Compared with well established reference databases, the gene expression profile in ARDS PMNs showed near-complete correlation to datasets derived from patients with sepsis and burns. Transcripts enriched in ARDS PMNs were differentially expressed in known functional network pathways associated with cancer, cellular compromise, apoptotic mechanisms, and chemotaxis. Of the observed gene changes, only 292 (22%) were seen in healthy volunteer PMNs after exposure to rhGM-CSF, of which 216 showed the same directional change as ARDS PMNs. INTERPRETATION Existing genome-wide studies in ARDS use total blood leucocytes; our study is the first, to our knowledge, to use unbiased global genomic profiling of highly pure ARDS blood PMNs in parallel with age-matched and gender-matched healthy volunteer PMNs treated with rhGM-CSF. Collectively our results show that ARDS PMNs display important de-novo transcriptional activity. The global transcriptomic changes were consistent with the observed aberrant ARDS PMN survival and functional phenotype that we have previously reported, and show near-complete correlation to existing sepsis and burns datasets, but only limited transcriptomic overlap with healthy volunteer PMNs treated with rhGM-CSF. FUNDING National Institute for Health Research, GlaxoSmithKline.

Functional capacity of alveolar neutrophils in acute respiratory distress syndrome

Abstract Background Inappropriate accumulation or persistence of neutrophils (polymorphonuclear leucocytes [PMNs]), within the alveolar airspace is considered to be crucial to the pathogenesis of acute respiratory distress syndrome (ARDS). Previous work has established that alveolar PMNs from patients with ARDS have a pro-survival phenotype, and that bronchoalveolar lavage fluid (BALF) in ARDS suppresses constitutive PMN apoptosis in vitro. However, the functional activity of alveolar-sequestered PMNs in ARDS is unknown, partly due to the technical challenges in obtaining and purifying alveolar PMNs. Proinflammatory mediators in the ARDS alveolar milieu conceivably prime or activate key PMN functions in vivo, including the generation of toxic reactive oxygen species (ROS). This research evaluates agonist-stimulated ROS generation in alveolar and blood PMNs from the same patient with ARDS compared with healthy volunteer blood PMNs. Methods Alveolar and blood PMNs from mechanically ventilated patients who fulfilled the Berlin criteria of ARDS were studied in parallel. Patients underwent fibreoptic bronchoscopy and bronchoalveolar lavage within 36 h of ARDS onset, and the recovered BALF was processed immediately. Alveolar PMNs were isolated to 90% (SE 2·5) purity with a rapid semi-automated negative-selection immunomagnetic technique (Robosep, Stemcell Technologies, Vancouver, Canada). BALF supernatants and serum were stored at −80°C for subsequent cytokine analysis. Patients undergoing bronchoalveolar lavage for other clinical indications (sarcoidosis, tracheomalacia, and haemoptysis) served as controls. Blood PMNs were isolated using dextran sedimentation and plasma Percoll gradients. Neutrophil apoptosis was detected by flow cytometry using annexin V and propidium iodide. Kinetics of the PMN oxidative burst in response to ex-vivo stimulation with N-formyl-methionyl-leucylphenylalanine was determined with real-time horseradish peroxidase–luminol-dependent chemiluminescence detection. Findings The BALF from patients with ARDS consisted predominantly of alveolar PMNs (65%, SE 5), whereas alveolar macrophages constituted the major cell type (92%, SE 5) in BALF from control individuals. Nearly 70% of ARDS alveolar PMNs showed hypersegmented nuclear morphology and had a reduced rate of apoptosis after 20 h in culture (22% apoptotic PMNs, SE 10) compared with blood PMNs from control individuals (68% apoptotic PMNs, SE 7·2). ROS production by untreated alveolar neutrophils was 1·5 times higher compared with optimally (tumour necrosis factor α) ex vivo-primed ARDS blood PMNs from the same ARDS patient and control blood PMNs. This finding could reflect the alveolar cytokine milieu since concentrations of the proinflammatory mediators interleukin (IL) 1, IL6, IL8, IL10, IL12, and tumour necrosis factor α were significantly increased in BALF and serum from patients with ARDS compared with controls. Interpretation These findings indicate that ARDS alveolar PMNs have altered morphology, enhanced survival, and augmented capacity for ROS generation, which might contribute to PMN-dependent tissue injury. Funding NIHR Cambridge Biomedical Research Centre, GlaxoSmithKline.