Tissue-Resident Alveolar Macrophages Reduce Ozone-induced Inflammation via MerTK-mediated Efferocytosis.

Lung inflammation, caused by acute exposure to ozone (O3), one of the six criteria air pollutants, is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung, and their number increases after O3 exposure. However, the role of AMØs in promoting or limiting O3-induced lung inflammation has not been clearly defined. In this study, we used a mouse model of acute O3 exposure, lineage tracing, genetic knockouts, and data from O3-exposed human volunteers to define the role and ontogeny of AMØs during acute O3 exposure. Lineage-tracing experiments showed that 12, 24, and 72 hours after exposure to O3 (2 ppm) for 3 hours, all AMØs were of tissue-resident origin. Similarly, in humans exposed to filtered air and O3 (200 ppb) for 135 minutes, we did not observe at ∼21 hours postexposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O3 exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØs demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK (MER proto-oncogene, tyrosine kinase), a key receptor involved in efferocytosis, also resulted in impaired clearance of apoptotic neutrophils after O3 exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis.

Allergic Asthma Responses Are Dependent on Macrophage Ontogeny.

The ontogenetic composition of tissue-resident macrophages following injury, environmental exposure, or experimental depletion can be altered upon re-establishment of homeostasis. However, the impact of altered resident macrophage ontogenetic milieu on subsequent immune responses is poorly understood. Hence, we assessed the effect of macrophage ontogeny alteration following return to homeostasis on subsequent allergic airway responses to house dust mites (HDM). Using lineage tracing, we confirmed alveolar and interstitial macrophage ontogeny and their replacement by bone marrow-derived macrophages following LPS exposure. This alteration in macrophage ontogenetic milieu reduced allergic airway responses to HDM challenge. In addition, we defined a distinct population of resident-derived interstitial macrophages expressing allergic airway disease genes, located adjacent to terminal bronchi, and reduced by prior LPS exposure. These findings support that the ontogenetic milieu of pulmonary macrophages is a central factor in allergic airway responses and has implications for how prior environmental exposures impact subsequent immune responses and the development of allergy.

Scavenger receptor BI attenuates oxidized phospholipid-induced pulmonary inflammation.

Damage associated molecular patterns (DAMPs) are molecules released from dead/dying cells following toxicant and/or environmental exposures that activate the immune response through binding of pattern recognition receptors (PRRs). Excessive production of DAMPs or failed clearance leads to chronic inflammation and delayed inflammation resolution. One category of DAMPs are oxidized phospholipids (oxPLs) produced upon exposure to high levels of oxidative stress, such as following ozone (O3) induced inflammation. OxPLs are bound by multiple classes of PRRs that include scavenger receptors (SRs) such as SR class B-1 (SR-BI) and toll-like receptors (TLRs). Interactions between oxPLs and PRRs appear to regulate inflammation; however, the role of SR-BI in oxPL-induced lung inflammation has not been defined. Therefore, we hypothesize that SR-BI is critical in protecting the lung from oxPL-induced pulmonary inflammation/injury. To test this hypothesis, C57BL/6J (WT) female mice were dosed with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (oxPAPC) by oropharyngeal aspiration which increased pulmonary SR-BI expression. Following oxPAPC exposure, SR-BI deficient (SR-BI-/-) mice exhibited increased lung pathology and inflammatory cytokine/chemokine production. Lipidomic analysis revealed that SR-BI-/- mice had an altered pulmonary lipidome prior to and following oxPAPC exposure, which correlated with increased oxidized phosphatidylcholines (PCs). Finally, we characterized TLR4-mediated activation of NF-κB following oxPAPC exposure and discovered that SR-BI-/- mice had increased TLR4 mRNA expression in lung tissue and macrophages, increased nuclear p65, and decreased cytoplasmic IκBα. Overall, we conclude that SR-BI is required for limiting oxPAPC-induced lung pathology by maintaining lipid homeostasis, reducing oxidized PCs, and attenuating TLR4-NF-κB activation, thereby preventing excessive and persistent inflammation.

Wood smoke particle exposure in mice reduces the severity of influenza infection.

Elevated ambient temperatures and extreme weather events have increased the incidence of wildfires world-wide resulting in increased wood smoke particle (WSP). Epidemiologic data suggests that WSP exposure associates with exacerbations of respiratory diseases, and with increased respiratory viral infections. To assess the impact of WSP exposure on host response to viral pneumonia, we performed WSP exposures in rodents followed by infection with mouse adapted influenza (HINI-PR8). C57BL/6 male mice aged 6-8 weeks were challenged with WSP or PBS by oropharyngeal aspiration in acute (single dose) or sub-acute exposures (day 1, 3, 5, 7 and 10). Additional groups underwent sub-acute exposure followed by infection by influenza or heat-inactivated (HI) virus. Following exposures/infection, bronchoalveolar lavage (BAL) was performed to assess for total cell counts/differentials, total protein, protein carbonyls and hyaluronan. Lung tissue was assessed for viral counts by real time PCR. When compared to PBS, acute WSP exposure associated with an increase in airspace macrophages. Alternatively, sub-acute exposure resulted in a dose dependent increase in airspace neutrophils. Sub-acute WSP exposure followed by influenza infection was associated with improved respiratory viral outcomes including reduced weight loss and increased blood oxygen saturation, and decreased protein carbonyls and viral titers. Flow cytometry demonstrated dynamic changes in pulmonary macrophage and T cell subsets based on challenge with WSP and influenza. This data suggests that sub-acute WSP exposure can improve host response to acute influenza infection.

Interleukin-13 disrupts type 2 pneumocyte stem cell activity.

The T helper 2 (Th2) inflammatory cytokine interleukin-13 (IL-13) has been associated with both obstructive and fibrotic lung diseases; however, its specific effect on the epithelial stem cells in the gas exchange compartment of the lung (alveolar space) has not been explored. Here, we used in vivo lung models of homeostasis and repair, ex vivo organoid platforms, and potentially novel quantitative proteomic techniques to show that IL-13 disrupts the self-renewal and differentiation of both murine and human type 2 alveolar epithelial cells (AEC2s). Significantly, we find that IL-13 promotes ectopic expression of markers typically associated with bronchiolar airway cells and commonly seen in the alveolar region of lung tissue from patients with idiopathic pulmonary fibrosis. Furthermore, we identify a number of proteins that are differentially secreted by AEC2s in response to IL-13 and may provide biomarkers to identify subsets of patients with pulmonary disease driven by "Th2-high" biology.

Silica Exposure Differentially Modulates Autoimmunity in Lupus Strains and Autoantibody Transgenic Mice.

Inhalational exposure to crystalline silica is linked to several debilitating systemic autoimmune diseases characterized by a prominent humoral immune component, but the mechanisms by which silica induces autoantibodies is poorly understood. To better understand how silica lung exposure breaks B cell tolerance and unleashes autoreactive B cells, we exposed both wildtype mice of healthy C57BL/6 and lupus-prone BXSB, MRL, and NZB strains and mice carrying an autoantibody transgene on each of these backgrounds to instilled silica or vehicle and monitored lung injury, autoimmunity, and B cell fate. Silica exposure induced lung damage and pulmonary lymphoid aggregates in all strains, including in genetically diverse backgrounds and in autoantibody transgenic models. In wildtype mice strain differences were observed in specificity of autoantibodies and site of enhanced autoantibody production, consistent with genetic modulation of the autoimmune response to silica. The unique autoantibody transgene reporter system permitted the in vivo fate of autoreactive B cells and tolerance mechanisms to be tracked directly, and demonstrated the presence of transgenic B cells and antibody in pulmonary lymphoid aggregates and bronchoalveolar lavage fluid, respectively, as well as in spleen and serum. Nonetheless, B cell enumeration and transgenic antibody quantitation indicated that B cell deletion and anergy were intact in the different genetic backgrounds. Thus, silica exposure sufficient to induce substantial lung immunopathology did not overtly disrupt central B cell tolerance, even when superimposed on autoimmune genetic susceptibility. This suggests that silica exposure subverts tolerance at alternative checkpoints, such as regulatory cells or follicle entry, or requires additional interactions or co-exposures to induce loss of tolerance. This possibility is supported by results of differentiation assays that demonstrated transgenic autoantibodies in supernatants of Toll-like receptor (TLR)7/TLR9-stimulated splenocytes harvested from silica-exposed, but not vehicle-exposed, C57BL/6 mice. This suggests that lung injury induced by silica exposure has systemic effects that subtly alter autoreactive B cell regulation, possibly modulating B cell anergy, and that can be unmasked by superimposed exposure to TLR ligands or other immunostimulants.

Immediate Release of Gastrin-Releasing Peptide Mediates Delayed Radiation-Induced Pulmonary Fibrosis.

Radiation-induced pulmonary fibrosis (RTPF) is a progressive, serious condition in many subjects treated for thoracic malignancies or after accidental nuclear exposure. No biomarker exists for identifying the irradiated subjects most susceptible to pulmonary fibrosis (PF). Previously, we determined that gastrin-releasing peptide (GRP) was elevated within days after birth in newborns exposed to hyperoxia who later developed chronic lung disease. The goal of the current study was to test whether radiation (RT) exposure triggers GRP release in mice and whether this contributes to RTPF in vivo. We determined urine GRP levels and lung GRP immunostaining in mice 0 to 24 after post-thoracic RT (15 Gy). Urine GRP levels were significantly elevated between 24 hours post-RT; GRP-blocking monoclonal antibody 2A11, given minutes post-RT, abrogated urine GRP levels by 6 to 12 hours and also altered phosphoprotein signaling pathways at 24 hours post-RT. Strong extracellular GRP immunostaining was observed in lung at 6 hours post-RT. Mice given one dose of GRP monoclonal antibody 2A11 24 hours post-RT had significantly reduced myofibroblast accumulation and collagen deposition 15 weeks later, indicating protection against lung fibrosis. Therefore, elevation of urine GRP could be predictive of RTPF development. In addition, transient GRP blockade could mitigate PF in normal lung after therapeutic or accidental RT exposure.

Sex Modifies Acute Ozone-Mediated Airway Physiologic Responses.

Sex differences clearly exist in incidence, susceptibility, and severity of airway disease and in pulmonary responses to air pollutants such as ozone (O3). Prior rodent O3 exposure studies demonstrate sex-related differences in the expression of lung inflammatory mediators and signaling. However, whether or not sex modifies O3-induced airway physiologic responses remains less explored. To address this, we exposed 8- to 10-week-old male and female C57BL/6 mice to either 1 or 2 ppm O3 or filtered air (FA) for 3 h. At 12, 24, 48, and 72 h following exposure, we assessed airway hyperresponsiveness to methacholine (MCh), bronchoalveolar lavage fluid cellularity, cytokines and total protein/albumin, serum progesterone, and whole lung immune cells by flow cytometry. Male mice generated consistent airway hyperresponsiveness to MCh at all time points following exposure. Alternatively, females had less consistent airway physiologic responses to MCh, which were more variable between individual experiments and did not correlate with serum progesterone levels. Bronchoalveolar lavage fluid total cells peaked at 12 h and were persistently elevated through 72 h. At 48 h, bronchoalveolar lavage cells were greater in females versus males. Bronchoalveolar lavage fluid cytokines and total protein/albumin increased following O3 exposure without sex differences. Flow cytometry of whole lung tissue identified dynamic O3-induced immune cell changes also independent of sex. Our results indicate sex differences in acute O3-induced airway physiology responses and airspace influx without significant difference in other injury and inflammation measures. This study highlights the importance of considering sex as a biological variable in acute O3-induced airway physiology responses.

Euthanasia- and Lavage-mediated Effects on Bronchoalveolar Measures of Lung Injury and Inflammation.

Accurate and reproducible assessments of experimental lung injury and inflammation are critical for basic and translational research. In particular, investigators use various methods for BAL and euthanasia; however, the impact of these methods on assessments of injury and inflammation is unknown. To define potential effects, we compared methods of lavage and euthanasia in uninjured mice and after a mild lung injury model (ozone). C57BL/6J male mice (8-10 weeks old) underwent BAL after euthanasia with ketamine/xylazine, carbon dioxide (CO2), or isoflurane. BAL methods included 800 µl of isotonic solution instilled and withdrawn three times, and one or three passive fills and drainage to 20 cm H2O. Parallel experiments were performed 24 hours after 3 hours of ozone (O3) exposure at 2 ppm. BAL total cell counts/differentials and total protein/albumin were determined. Lung histology was evaluated for lung inflammation or injury. BAL cells were cultured and stimulated with PBS, PMA, or LPS for 4 hours and supernatants were evaluated for cytokine content. In uninjured mice, we observed differences due to the lavage and euthanasia methods used. The lavage method increased total cells and total protein/albumin in uninjured and O3-exposed mice, with the 800-µl instillation having the highest values. Isoflurane increased total BAL cells, whereas CO2 euthanasia increased the total protein/albumin levels in uninjured mice. These effects limited our ability to detect differences in BAL injury measures after O3 exposure. In conclusion, the method used for lavage and euthanasia affects measures of lung inflammation/injury and should be considered a variable in model assessments.

Arginase1 Deficiency in Monocytes/Macrophages Upregulates Inducible Nitric Oxide Synthase To Promote Cutaneous Contact Hypersensitivity.

The innate immune components that modulate allergic contact hypersensitivity (CHS) responses are poorly defined. Using human skin from contact dermatitis patients and a mouse model of CHS, we find that hapten allergens disrupt the Arginase1 (Arg1) and inducible NO synthase (iNOS) dynamic in monocytes/macrophages (mono/MΦ), which renders those cells ineffectual in suppressing skin inflammation. Mice lacking Arg1 in MΦ develop increased CHS characterized by elevated ear thickening, mono/MΦ-dominated dermal inflammation, and increased iNOS and IL-6 expression compared with control mice. Treatment of Arg1flox/flox; LysMCre+/- mice with a selective NOS inhibitor or knockout of Nos2, encoding iNOS, significantly ameliorates CHS. Our findings suggest a critical role for Arg1 in mono/MΦ in suppressing CHS through dampening Nos2 expression. These results support that increasing Arg1 may be a potential therapeutic avenue in treating allergic contact dermatitis.

Full Spectrum of LPS Activation in Alveolar Macrophages of Healthy Volunteers by Whole Transcriptomic Profiling.

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-ß (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/ß 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.

Modulating distal cavities in the α and ß subunits of human HbA reveals the primary ligand migration pathway.

The free volume in the active site of human HbA plays a crucial role in governing the bimolecular rates of O(2), CO, and NO binding, the fraction of geminate ligand recombination, and the rate of NO dioxygenation by the oxygenated complex. We have decreased the size of the distal pocket by mutating Leu(B10), Val(E11), and Leu(G8) to Phe and Trp and that of other more internal cavities by filling them with Xe at high gas pressures. Increasing the size of the B10 side chain reduces bimolecular rates of ligand binding nearly 5000-fold and inhibits CO geminate recombination due to both reduction of the capture volume in the distal pocket and direct steric hindrance of Fe-ligand bond formation. Phe and Trp(E11) mutations also cause a decrease in distal pocket volume but, at the same time, increase access to the Fe atom because of the loss of the γ2 CH(3) group of the native Val(E11) side chain. The net result of these E11 substitutions is a dramatic increase in the rate of geminate recombination because dissociated CO is sequestered close to the Fe atom and can rapidly rebind without steric resistance. However, the bimolecular rate constants for binding of ligand to the Phe and Trp(E11) mutants are decreased 5-30-fold, because of a smaller capture volume. Geminate and bimolecular kinetic parameters for Phe and Trp(G8) mutants are similar to those for the native HbA subunits because the aromatic rings at this position cause little change in distal pocket volume and because ligands do not move past this position into the globin interior of wild-type HbA subunits. The latter conclusion is verified by the observation that Xe binding to the α and ß Hb subunits has little effect on either geminate or bimolecular ligand rebinding. All of these experimental results argue strongly against alternative ligand migration pathways that involve movements through the protein interior in HbA. Instead, ligands appear to enter through the His(E7) gate and are captured directly in the distal cavity.