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Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry–based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206+) subsets and other CD206− leukocytes. The CD206− cells include: (1) three monocyte (CD14+) subsets, (2) CD11c+ dendritic cells (CD14−, CD11c+, HLA-DR+), (3) plasmacytoid dendritic cells (CD14−, CD11c−, HLA-DR+, CD123+), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169+ and CD169− macrophages. In lung tissue, CD169− macrophages were a prominent cell type. Using confocal microscopy, CD169+ macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169− macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples.

Although the antibody-based recognition of cell-surface markers has been widely used for the identification of immune cells, overlap in the expression of markers by different cell types and the inconsistent use of antibody panels have resulted in a lack of clearly defined signatures for myeloid cell subsets. We developed a 10-fluorochrome flow cytometry panel for the identification and quantitation of myeloid cells in the lungs, including pulmonary monocytes, myeloid dendritic cells, alveolar and interstitial macrophages, and neutrophils. After the initial sorting of viable CD45+ leukocytes, we detected three leukocyte subpopulations based on CD68 expression: CD68−, CD68low, and CD68hi. Further characterization of the CD68hi population revealed CD45+/CD68hi/F4/80+/CD11b−/CD11c+/Gr1− alveolar macrophages and CD45+/CD68hi/F4/80−/CD11c+/Gr1−/CD103+/major histocompatibility complex (MHC) class IIhi dendritic cells. The CD68low population contained primarily CD45+/CD68low/F4/80+/CD11b+/CD11c+/Gr1−/CD14low interstitial macrophages and CD45+/CD68low/F4/80+/CD11b+/CD11c−/Gr1low/CD14hi monocytes, whereas the CD68− population contained neutrophils (CD45+/CD68−/F4/80−/CD11b+/Gr1hi). The validity of cellular signatures was confirmed by a morphological analysis of FACS-sorted cells, functional studies, and the depletion of specific macrophage subpopulations using liposomal clodronate. We believe our approach provides an accurate and reproducible method for the isolation, quantification, and characterization of myeloid cell subsets in the lungs, which may be useful for studying the roles of myeloid cells during various pathological processes.

Background Microbial dysbiosis is a potential mediator of air pollution-induced adverse outcomes. However, a systemic comparison of the lung and gut microbiome alterations and lung-gut axis following air pollution exposure is scant. In this study, we exposed male C57BL/6J mice to inhaled air, CB (10 mg/m 3 ), O 3 (2 ppm) or CB + O 3 mixture for 3 h/day for either one day or four consecutive days and were euthanized 24 h post last exposure. The lung and gut microbiome were quantified by 16 s sequencing. Results Multiple CB + O 3 exposures induced an increase in the lung inflammatory cells (neutrophils, eosinophils and B lymphocytes), reduced absolute bacterial load in the lungs and increased load in the gut. CB + O 3 exposure was more potent as it decreased lung microbiome alpha diversity just after a single exposure. CB + O 3 co-exposure uniquely increased Clostridiaceae and Prevotellaceae in the lungs. Serum short chain fatty acids (SCFA) (acetate and propionate) were increased significantly only after CB + O 3 co-exposure. A significant increase in SCFA producing bacterial families ( Ruminococcaceae , Lachnospiraceae , and Eubacterium ) were also observed in the gut after multiple exposures. Co-exposure induced significant alterations in the gut derived metabolite receptors/mediator ( Gcg, Glp-1r, Cck ) mRNA expression. Oxidative stress related mRNA expression in lungs, and oxidant levels in the BALF, serum and gut significantly increased after CB + O 3 exposures. Conclusion Our study confirms distinct gut and lung microbiome alterations after CB + O 3 inhalation co-exposure and indicate a potential homeostatic shift in the gut microbiome to counter deleterious impacts of environmental exposures on metabolic system.

The epidemiology of Interstitial Lung Diseases (ILD) in the Veterans Health Administration (VHA) is presently unknown. Describe the incidence/prevalence, clinical characteristics, and outcomes of ILD patients within the Veteran's Administration Mid-Atlantic Health Care Network (VISN6). A multi-center retrospective cohort study was performed of veterans receiving hospital or outpatient ILD care from January 1, 2008 to December 31st, 2015 in six VISN6 facilities. Patients were identified by at least one visit encounter with a 515, 516, or other ILD ICD-9 code. Demographic and clinical characteristics were summarized using median, 25th and 75th percentile for continuous variables and count/percentage for categorical variables. Characteristics and incidence/prevalence rates were summarized, and stratified by ILD ICD-9 code. Kaplan Meier curves were generated to define overall survival. 3293 subjects met the inclusion criteria. 879 subjects (26%) had no evidence of ILD following manual medical record review. Overall estimated prevalence in verified ILD subjects was 256 per 100,000 people with a mean incidence across the years of 70 per 100,000 person-years (0.07%). The prevalence and mean incidence when focusing on people with an ILD diagnostic code who had a HRCT scan or a bronchoscopic or surgical lung biopsy was 237 per 100,000 people (0.237%) and 63 per 100,000 person-years respectively (0.063%). The median survival was 76.9 months for 515 codes, 103.4 months for 516 codes, and 83.6 months for 516.31. This retrospective cohort study defines high ILD incidence/prevalence within the VA. Therefore, ILD is an important VA health concern.

Occupational exposure to inhaled crystalline silica dust (cSiO2) is linked to systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, and anti-neutrophil cytoplasmic autoantibody vasculitis. Each disease has a characteristic autoantibody profile used in diagnosis and implicated in pathogenesis. A role for cSiO2 in modulating humoral autoimmunity in vivo is supported by findings in mice, where respirable cSiO2 induces ectopic lymphoid structures as well as inflammation in exposed lungs across genetically diverse backgrounds. In lupus-prone mice cSiO2 exposure also leads to early onset autoantibody production and accelerated disease. Elevated autoantibody levels in bronchoalveolar lavage fluid (BALF) and lung transcriptome analysis suggest that the lung is a hub of cSiO2-evoked autoimmune activity. However, mechanisms by which cSiO2 and lung microenvironments interact to promote autoantibody production remain unclear. We previously demonstrated elevated anti-DNA Ig in BALF but not in lung cell cultures from cSiO2-exposed C57BL/6 mice, suggesting that BALF autoantibodies did not arise locally in this non-autoimmune strain. Autoantibodies were also elevated in BALF of cSiO2-exposed lupus-prone BXSB mice. In this report we test the hypothesis that dysregulated autoreactive B cells recruited to cSiO2-exposed lungs in the context of autoimmune predisposition contribute to local autoantibody production. We found that anti-DNA and anti-myeloperoxidase (MPO) Ig were significantly elevated in cultures of TLR ligand-stimulated lung cells from cSiO2-exposed BXSB mice. To further explore the impact of strain genetic susceptibility versus B cell intrinsic dysfunction on cSiO2-recruited B cell fate, we used an anti-basement membrane autoantibody transgenic (autoAb Tg) mouse line termed M7. In M7 mice, autoAb Tg B cells are aberrantly regulated and escape from tolerance on the C57BL/6 background. Exposure to cSiO2 elicited prominent pulmonary B cell and T cell aggregates and autoAb Tg Ig were readily detected in lung cell culture supernatants. Taken together, diverse disease-relevant autoreactive B cells, including cells specific for DNA, MPO, and basement membrane, are recruited to lung ectopic lymphoid aggregates in response to cSiO2 instillation. B cells that escape tolerance can contribute to local autoantibody production. Our demonstration of significantly enhanced autoantibody induction by TLR ligands further suggests that a coordinated environmental co-exposure can magnify autoimmune vulnerability.

Although IL-9 has potent anti-tumor activity in adoptive cell transfer therapy, some models suggest that it can promote tumor growth. Here, we show that IL-9 signaling is associated with poor outcomes in patients with various forms of lung cancer, and is required for lung tumor growth in multiple mouse models. CD4 + T cell-derived IL-9 promotes the expansion of both CD11c + and CD11c − interstitial macrophage populations in lung tumor models. Mechanistically, the IL-9/macrophage axis requires arginase 1 (Arg1) to mediate tumor growth. Indeed, adoptive transfer of Arg1 + but not Arg1 - lung macrophages to Il9r −/− mice promotes tumor growth. Moreover, targeting IL-9 signaling using macrophage-specific nanoparticles restricts lung tumor growth in mice. Lastly, elevated expression of IL-9R and Arg1 in tumor lesions is associated with poor prognosis in lung cancer patients. Thus, our study suggests the IL-9/macrophage/Arg1 axis is a potential therapeutic target for lung cancer therapy. The role of IL-9 in the tumor microenvironment and its effects on macrophages remains unclear. Here, the authors show that IL-9 promotes the expansion of pulmonary macrophages and that targeting the IL-9R/arginase 1 axis restricts tumor growth, thus identifying this cytokine pathway as a potential therapeutic target.

Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, for example through lipopolysaccharide (LPS) or ozone (O 3 ). Toll-like receptors (TLRs) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O 3 , triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O 3 , hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O 3 . We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O 3 exposure ex vivo and LPS exposure in vitro. Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling. Immune cells in the lung help guard against infections. On the surface of these cells are proteins called TLR receptors that recognize dangerous molecules or DNA from disease-causing microbes such as bacteria. When the immune cells detect these invaders, the TLR receptors spring into action and trigger an inflammatory response to destroy the microbes. This inflammation usually helps the lung clear infections. But it can also be harmful and damage the lung, for example when inflammation is caused by non-infectious substances such as pollutants in the atmosphere. There are several TLR receptors that each recognize a specific molecule. In 2010, researchers showed that the receptor TLR4 is responsible for causing inflammation in the lung after exposure to pollution. Another receptor called TLR5 also helps activate the immune response in the lung. But it was unclear whether this receptor also plays a role in pollution-linked lung damage. Now, Hussain, Johnson, Sciurba et al. – including one of the researchers involved in the 2010 study – have investigated the role of TLR5 in immune cells from the lungs of humans and mice. The experiments showed that TLR5 works together with TLR4 and helps trigger an inflammatory response to both pollutants and bacteria. Hussain et al. found that people lacking a working TLR5 receptor (which make up 3–10% of the population) are less likely to experience lung inflammation when exposed to pollution or bacterial proteins that activate TLR4. These findings suggest that people without TLR5 may be protected from pollution-induced lung injury. Further research into the role of TLR5 could help develop genetic tests for identifying people who are more sensitive to damage from pollution. This information could then be used to determine the likelihood of a patient experiencing certain lung diseases.

Increased exposure to Ozone (O3) is associated with adverse health effects in individuals afflicted with respiratory diseases. Surfactant protein-A (SP-A), encoded by SP-A1 and SP-A2, is the largest protein component in pulmonary surfactant and is functionally impaired by O3-oxidation. We used humanized SP-A2 transgenic mice with allelic variation corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 in the lectin domain to determine the impact of this genetic variation in regards to O3 exposure. Mice were exposed to 2ppm O3 or Filtered Air (FA) for 3 hours and 24 hrs post-challenge pulmonary function tests and other parameters associated with inflammation were assessed in the bronchoalveolar lavage (BAL) fluid and lung tissue. Additionally, mouse tracheal epithelial cells were cultured and TEER measurements recorded for each genotype to determine baseline epithelial integrity. Compared to FA, O3 exposure led to significantly increased sensitivity to methacholine challenge in all groups of mice. SP-A2 223Q variant mice were significantly protected from O3-induced AHR compared to SP-A-/- and SP-A2 223K mice. Neutrophilia was observed in all genotypes of mice post O3-exposure, however, SP-A2 223Q mice had a significantly lower percentage of neutrophils compared to SP-A-/- mice. Albumin levels in BAL were unchanged in O3-exposed SP-A2 223Q mice compared to their FA controls, while levels were significantly increased in all other genotypes of O3-exposed mice. SP-A 223Q MTECS has significant higher TEER values than all other genotypes, and WT MTECS has significantly higher TEER than the SP-A KO and SP-A 223K MTECS. Taken together, our study suggests that expression of a glutamine (Q) as position 223 in SP-A2, as opposed to expression of lysine (K), is more protective in acute exposures to ozone and results in attenuated O3-induced AHR, neutrophilia, and vascular permeability.

Despite recent advances in understanding macrophage activation, little is known regarding how human alveolar macrophages in health calibrate its transcriptional response to canonical TLR4 activation. In this study, we examined the full spectrum of LPS activation and determined whether the transcriptomic profile of human alveolar macrophages is distinguished by a TIR-domain-containing adapter-inducing interferon-β (TRIF)-dominant type I interferon signature. Bronchoalveolar lavage macrophages were obtained from healthy volunteers, stimulated in the presence or absence of ultrapure LPS in vitro, and whole transcriptomic profiling was performed by RNA sequencing (RNA-Seq). LPS induced a robust type I interferon transcriptional response and Ingenuity Pathway Analysis predicted interferon regulatory factor (IRF)7 as the top upstream regulator of 89 known gene targets. Ubiquitin-specific peptidase (USP)-18, a negative regulator of interferon α/β responses, was among the top up-regulated genes in addition to IL10 and USP41, a novel gene with no known biological function but with high sequence homology to USP18. We determined whether IRF-7 and USP-18 can influence downstream macrophage effector cytokine production such as IL-10. We show that IRF-7 siRNA knockdown enhanced LPS-induced IL-10 production in human monocyte-derived macrophages, and USP-18 overexpression attenuated LPS-induced production of IL-10 in RAW264.7 cells. Quantitative PCR confirmed upregulation of USP18, USP41, IL10, and IRF7. An independent cohort confirmed LPS induction of USP41 and IL10 genes. These results suggest that IRF-7 and predicted downstream target USP18, both elements of a type I interferon gene signature identified by RNA-Seq, may serve to fine-tune early cytokine response by calibrating IL-10 production in human alveolar macrophages.

Background Interactions between air pollution and infectious agents are increasingly recognized and critical to identify, especially to protect vulnerable populations. Pregnancy represents a vulnerable period for influenza infection and air pollution exposure, yet interactions during pregnancy remain unclear. Maternal exposure to ultrafine particles (UFPs, ≤ 100 nm diameter), a class of particulate matter ubiquitous in urban environments, elicits unique pulmonary immune responses. We hypothesized that UFP exposure during pregnancy would lead to aberrant immune responses to influenza enhancing infection severity. Results Building from our well-characterized C57Bl/6N mouse model employing daily gestational UFP exposure from gestational day (GD) 0.5–13.5, we carried out a pilot study wherein pregnant dams were subsequently infected with Influenza A/Puerto Rico/8/1934 (PR8) on GD14.5. Findings indicate that PR8 infection caused decreased weight gain in filtered air (FA) and UFP-exposed groups. Co-exposure to UFPs and viral infection led to pronounced elevation in PR8 viral titer and reduced pulmonary inflammation, signifying potential suppression of innate and adaptive immune defenses. Pulmonary expression of the pro-viral factor sphingosine kinase 1 ( Sphk1 ) and pro-inflammatory cytokine interleukin-1β ( IL-1 β ) was significantly increased in pregnant mice exposed to UFPs and infected with PR8; expression correlated with higher viral titer. Conclusions Results from our model provide initial insight into how maternal UFP exposure during pregnancy enhances respiratory viral infection risk. This model is an important first step in establishing future regulatory and clinical strategies for protecting pregnant women exposed to UFPs.