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Background Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a “just-in-time” design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument. Methods The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data. Results Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel. Conclusions Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters. Trial registration This study was registered with ClinicalTrials.gov as NCT01969344 .

Background Cigarette smoking is associated with increased frequency and duration of viral respiratory infections, but the underlying mechanisms are incompletely defined. We investigated whether smoking reduces expression by human lung macrophages (Mø) of receptors for viral nucleic acids and, if so, the effect on CXCL10 production. Methods We collected alveolar macrophages (AMø) by bronchoalveolar lavage of radiographically-normal lungs of subjects undergoing bronchoscopies for solitary nodules ( n  = 16) and of volunteers who were current or former smokers ( n  = 7) or never-smokers ( n  = 13). We measured expression of mRNA transcripts for viral nucleic acid receptors by real-time PCR in those AMø and in the human Mø cell line THP-1 following phorbol myristate acetate/vitamin D3 differentiation and exposure to cigarette smoke extract, and determined TLR3 protein expression using flow cytometry and immunohistochemistry. We also used flow cytometry to examine TLR3 expression in total lung Mø from subjects undergoing clinically-indicated lung resections ( n  = 25). Of these, seven had normal FEV1 and FEV1/FVC ratio (three former smokers, four current smokers); the remaining 18 subjects (14 former smokers; four current smokers) had COPD of GOLD stages I-IV. We measured AMø production of CXCL10 in response to stimulation with the dsRNA analogue poly(I:C) using Luminex assay. Results Relative to AMø of never-smokers, AMø of smokers demonstrated reduced protein expression of TLR3 and decreased mRNA for TLR3 but not TLR7, TLR8, TLR9, RIG-I, MDA-5 or PKR. Identical changes in TLR3 gene expression were induced in differentiated THP-1 cells exposed to cigarette smoke-extract in vitro for 4 hours. Among total lung Mø, the percentage of TLR3-positive cells correlated inversely with active smoking but not with COPD diagnosis, FEV1% predicted, sex, age or pack-years. Compared to AMø of never-smokers, poly(I:C)-stimulated production of CXCL10 was significantly reduced in AMø of smokers. Conclusions Active smoking, independent of COPD stage or smoking duration, reduces both the percent of human lung Mø expressing TLR3, and dsRNA-induced CXCL10 production, without altering other endosomal or cytoplasmic receptors for microbial nucleic acids. This effect provides one possible mechanism for increased frequency and duration of viral lower respiratory tract infections in smokers. Trial registration ClinicalTrials.gov NCT00281190 , NCT00281203 and NCT00281229 .

Innate immune responses are essential for controlling poxvirus infection. The threat of a bioterrorist attack using Variola major, the smallpox virus, or zoonotic transmission of other poxviruses has renewed interest in understanding interactions between these viruses and their hosts. We recently determined that TLR3 regulates a detrimental innate immune response that enhances replication, morbidity, and mortality in mice in response to vaccinia virus, a model pathogen for studies of poxviruses. To further investigate Toll-like receptor signaling in vaccinia infection, we first focused on TRIF, the only known adapter protein for TLR3. Unexpectedly, bioluminescence imaging showed that mice lacking TRIF are more susceptible to vaccinia infection than wild-type mice. We then focused on TLR4, the other Toll-like receptor that signals through TRIF. Following respiratory infection with vaccinia, mice lacking TLR4 signaling had greater viral replication, hypothermia, and mortality than control animals. The mechanism of TLR4-mediated protection was not due to increased release of proinflammatory cytokines or changes in total numbers of immune cells recruited to the lung. Challenge of primary bone marrow macrophages isolated from TLR4 mutant and control mice suggested that TLR4 recognizes a viral ligand rather than an endogenous ligand. These data establish that TLR4 mediates a protective innate immune response against vaccinia virus, which informs development of new vaccines and therapeutic agents targeted against poxviruses.

Abstract Apoptotic cells must be cleared efficiently by macrophages (Mø) to prevent autoimmunity, yet their ingestion impairs Mø microbicidal function. The principal murine resident lung phagocyte, the alveolar Mø (AMø), is specifically deficient at apoptotic cell ingestion, both in vitro and in vivo, compared with resident peritoneal Mø (PMø). To further characterize this deficiency, we assayed static adhesion in vitro using apoptotic thymocytes and resident AMø and PMø from normal C57BL/6 mice. Adhesion of apoptotic thymocytes by both types of Mø was rapid, specific, and cold-sensitive. Antibody against the receptor tyrosine kinase MerTK (Tyro12) blocked phagocytosis but not adhesion in both types of Mø. Surfactant protein A increased adhesion and phagocytosis by AMø, but not to the levels seen using PMø. Adhesion was largely cation-independent for PMø and calcium-dependent for AMø. Adhesion was not inhibited in either Mø type by mAbs against β1 or β3 integrins or scavenger receptor I/II (CD204), but AMø adhesion was inhibited by specific mAbs against CD11c/CD18. Thus, resident murine tissue Mø from different tissues depend on qualitatively disparate receptor systems to bind apoptotic cells. The decreased capacity of murine AMø to ingest apoptotic cells is only partially explained by reduced initial adhesion.

Mice with targeted mutation of chemokine receptor 1 (CCR1) were used to assess the contribution of CCR1 agonists to local, regional, and systemic inflammatory-related events during experimental pulmonary granuloma formation. Models of Th1 (type-1) and Th2 (type-2) cell-mediated lung granulomas were induced in wild-type (CCR+/+) and knockout (CCR1−/−) mice by embolizing Sepharose beads coupled to the purified protein derivative of Mycobacterium bovis or soluble antigens derived from Schistosoma mansoni eggs. Morphometric analysis indicated that granuloma sizes were unchanged in CCR1−/− mice, but flow cytometric analyses of dispersed granulomas revealed that natural killer cell recruitment to type-1 lesions was abrogated by 60%. Analysis of cytokine production by draining lymph node cultures showed altered expression in CCR1−/− mice characterized by reduced interleukin-2 and interferon-γ in the type-1 response, and enhanced interleukin-5 and interleukin-13 in the type-2 response. Peripheral blood leukocytosis was also enhanced in the type-1 but not the type-2 response. These findings suggest that CCR1 agonists contribute to multiple immunoinflammatory events in the type-1 granulomatous response with natural killer cell accumulation being particularly sensitive to CCR1 disruption. Although functional efficacy of granulomas may be altered, chemokine redundancy and cytokine reserve seem to make the bulk of the exudative response resistant to CCR1 disruption.