Carla M. T. Bauer

Self-renewing resident arterial macrophages arise from embryonic CX3CR1+ precursors and circulating monocytes immediately after birth

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1+ precursors and postnatally from bone marrow–derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.

IL-1α/IL-1R1 Expression in Chronic Obstructive Pulmonary Disease and Mechanistic Relevance to Smoke-Induced Neutrophilia in Mice

Background Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. Despite this, the cellular and molecular mechanisms that contribute to COPD pathogenesis are still poorly understood. Methodology and Principal Findings The objective of this study was to assess IL-1 α and β expression in COPD patients and to investigate their respective roles in perpetuating cigarette smoke-induced inflammation. Functional studies were pursued in smoke-exposed mice using gene-deficient animals, as well as blocking antibodies for IL-1α and β. Here, we demonstrate an underappreciated role for IL-1α expression in COPD. While a strong correlation existed between IL-1α and β levels in patients during stable disease and periods of exacerbation, neutrophilic inflammation was shown to be IL-1α-dependent, and IL-1β- and caspase-1-independent in a murine model of cigarette smoke exposure. As IL-1α was predominantly expressed by hematopoietic cells in COPD patients and in mice exposed to cigarette smoke, studies pursued in bone marrow chimeric mice demonstrated that the crosstalk between IL-1α+ hematopoietic cells and the IL-1R1+ epithelial cells regulates smoke-induced inflammation. IL-1α/IL-1R1-dependent activation of the airway epithelium also led to exacerbated inflammatory responses in H1N1 influenza virus infected smoke-exposed mice, a previously reported model of COPD exacerbation. Conclusions and Significance This study provides compelling evidence that IL-1α is central to the initiation of smoke-induced neutrophilic inflammation and suggests that IL-1α/IL-1R1 targeted therapies may be relevant for limiting inflammation and exacerbations in COPD.

Bleomycin induces molecular changes directly relevant to idiopathic pulmonary fibrosis: a model for "active" disease.

The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFβ was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.

An Accessory to the ‘Trinity’: SR-As Are Essential Pathogen Sensors of Extracellular dsRNA, Mediating Entry and Leading to Subsequent Type I IFN Responses

Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (ds)RNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN) responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As) are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNβ. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG) induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as ‘carriers’, facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.

Innate immune processes are sufficient for driving cigarette smoke-induced inflammation in mice.

The objective of this study was to characterize the impact of cigarette smoke exposure on lung immune and inflammatory processes. BALB/c and C57BL/6 mice were exposed to cigarette smoke for 4 days (acute) or at least 5 weeks (prolonged). Both mouse strains manifested an inflammatory response after acute smoke exposure, characterized by an influx of neutrophils and mononuclear cells. Multiplex analysis revealed a greater than twofold increase of the cytokines IL-1alpha, -5, -6, and -18, as well as the chemokines monocyte chemotactic protein-1 and -3, macrophage inflammatory protein-1alpha, -beta, and -gamma, -2, -3beta, macrophage defined chemokine, granulocyte chemotactic protein-2, and interferon-gamma-inducible protein-10. In BALB/c mice, neutrophilia persisted after prolonged exposure, whereas C57BL/6 showed evidence of attenuated neutrophilia both in the bronchoalveolar lavage and the lungs. In both mouse strains, cigarette smoke exposure was associated with an expansion of mature (CD11c(hi)/major histocompatibility complex class II(hi)) myeloid dendritic cells; we observed no changes in plasmacytoid dendritic cells. Lymphocytes in the lungs displayed an activated phenotype that persisted for CD4 T cells only after prolonged exposure. In BALB/c mice, T cells acquired T helper (Th) 1 and Th2 effector function after 5 weeks of smoke exposure, whereas, in C57BL/6 mice, neither Th1 nor Th2 cells were detected. In both mouse strains, cigarette smoke exposure led to an accumulation of FoxP3+ T regulatory cells in the lungs. Studies in RAG1 knockout mice suggest that these regulatory cells may participate in controlling smoke-induced inflammation. Acute and prolonged cigarette smoke exposure was associated with inflammation, activation of the adaptive immune system, and expansion of T regulatory cells in the lungs.

Treating Viral Exacerbations of Chronic Obstructive Pulmonary Disease: Insights from a Mouse Model of Cigarette Smoke and H1N1 Influenza Infection

Background Chronic obstructive pulmonary disease is a progressive lung disease that is punctuated by periods of exacerbations (worsening of symptoms) that are attributable to viral infections. While rhinoviruses are most commonly isolated viruses during episodes of exacerbation, influenza viruses have the potential to become even more problematic with the increased likelihood of an epidemic. Methodology and Principal Findings This study examined the impact of current and potential pharmacological targets namely the systemic corticosteroid dexamethasone and the peroxisome proliferator-activated receptor- gamma agonist pioglitazone on the outcome of infection in smoke-exposed mice. C57BL/6 mice were exposed to room air or cigarette smoke for 4 days and subsequently inoculated with an H1N1 influenza A virus. Interventions were delivered daily during the course of infection. We show that smoke-exposed mice have an exacerbated inflammatory response following infection. While smoke exposure did not compromise viral clearance, precision cut lung slices from smoke-exposed mice showed greater expression of CC (MCP-1, -3), and CXC (KC, MIP-2, GCP-2) chemokines compared to controls when stimulated with a viral mimic or influenza A virus. While dexamethasone treatment partially attenuated the inflammatory response in the broncho-alveolar lavage of smoke-exposed, virally-infected animals, viral-induced neutrophilia was steroid insensitive. In contrast to controls, dexamethasone-treated smoke-exposed influenza-infected mice had a worsened health status. Pioglitazone treatment of virally-infected smoke-exposed mice proved more efficacious than the steroid intervention. Further mechanistic evaluation revealed that a deficiency in CCR2 did not improve the inflammatory outcome in smoke-exposed, virally-infected animals. Conclusions and Significance This animal model of cigarette smoke and H1N1 influenza infection demonstrates that smoke-exposed animals are differentially primed to respond to viral insult. While providing a platform to test pharmacological interventions, this model demonstrates that treating viral exacerbations with alternative anti-inflammatory drugs, such as PPAR-gamma agonists should be further explored since they showed greater efficacy than systemic corticosteroids.

Adjuvant and Anti-Inflammatory Properties of Cigarette Smoke in Murine Allergic Airway Inflammation

The impact of cigarette smoke on allergic asthma remains controversial both clinically and experimentally. The objective of this study was to investigate, in a murine model, how cigarette smoke affects immune inflammatory processes elicited by a surrogate allergen. In our experimental design, mice were concurrently exposed to cigarette smoke and ovalbumin (OVA), an innocuous antigen that, unless introduced in the context of an adjuvant, induces inhalation tolerance. We show that cigarette smoke exposure has adjuvant properties, allowing for allergic mucosal sensitization to OVA. Specifically, concurrent exposure to cigarette smoke and OVA for 2 weeks led to airway eosinophilia and goblet cell hyperplasia. In vivo OVA recall challenge 1 month after the last smoke exposure showed that concurrent exposure to OVA and cigarette smoke induced antigen-specific memory. Robust eosinophilia and OVA-specific IgG1 and IgE characterized the ensuing inflammatory response. Mechanistically, allergic sensitization was, in part, granulocyte macrophage colony-stimulating factor (GM-CSF) dependent, as a significant reduction in BAL eosinophilia was observed in mice treated with an anti-GM-CSF antibody. Of note, continuous smoke exposure attenuated the OVA recall response; decreased airway eosinophilia was observed in mice continuously exposed to cigarette smoke compared with mice that ceased the smoke exposure protocol. In conclusion, we demonstrate experimentally that while cigarette smoke acts as an adjuvant allowing for allergic sensitization, it also attenuates the ensuing eosinophilic inflammatory response.

Cigarette smoke suppresses type I interferon-mediated antiviral immunity in lung fibroblast and epithelial cells.

The objective of this study was to investigate the impact of cigarette smoke on innate antiviral defense mechanisms; specifically, we examined the effects of cigarette smoke on the induction of type I interferon (IFN). We observed a dose-dependent decrease in the ability of human lung fibroblast and epithelial cells to elicit an antiviral response against a viral double-strand RNA (dsRNA) mimic, polyI:C, in the presence of cigarette smoke-conditioned medium (SCM). Mechanistically, SCM decreases the expression of IFN-stimulated gene 15 (ISG15) and IFN regulatory factor-7 (IRF-7) transcripts and suppresses the nuclear translocation of key transcription factors, nuclear factor-kappaB (NF-kappaB) and IRF-3, after polyI:C stimulation. Furthermore, we provide evidence that the intercellular defense strategy against viral infection is also impaired. We observed a decrease in the ability of fibroblasts to elicit an antiviral state in response to IFN-beta stimulation. This was associated with decreased nuclear translocation of phosphorylated Stat1 in response to IFN-beta treatment. The effects elicited by SCM are reversible and are almost entirely abrogated in the presence of an antioxidant, such as glutathione. Our findings suggest that cigarette smoke affects the immediate-early, inductive, and amplification phases of the type I IFN response.

Interleukin-17A Promotes Neutrophilia in Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

RATIONALE Nontypeable Haemophilus influenzae (NTHi) causes acute exacerbation of chronic obstructive pulmonary disease (AECOPD). IL-17A is central for neutrophilic inflammation and has been linked to COPD pathogenesis. OBJECTIVES We investigated whether IL-17A is elevated in NTHi-associated AECOPD and required for NTHi-exacerbated pulmonary neutrophilia induced by cigarette smoke. METHODS Experimental studies with cigarette smoke and NTHi infection were pursued in gene-targeted mice and using antibody intervention. IL-17A was measured in sputum collected from patients with COPD at baseline, during, and after AECOPD. MEASUREMENTS AND MAIN RESULTS Exacerbated airway neutrophilia in cigarette smoke-exposed mice infected with NTHi was associated with an induction of IL-17A. In agreement, elevated IL-17A was observed in sputum collected during NTHi-associated AECOPD, compared with samples collected before or after the event. NTHi-exacerbated neutrophilia and induction of neutrophil chemoattractants over the background of cigarette smoke, as observed in wild-type mice, was absent in Il17a(-/-) mice and in mice treated with a neutralizing anti-IL-17A antibody. Further studies revealed that IL-1 receptor (R)1 signaling was required for IL-17A-dependent neutrophilia. Moreover, deficiency or therapeutic neutralization of IL-17A did not increase bacterial burden or delay bacterial clearance. CONCLUSIONS IL-17A is induced during NTHi-associated AECOPD. Functionally, IL-1R1-dependent IL-17A is required for NTHi-exacerbated pulmonary neutrophilia induced by cigarette smoke. Targeting IL-17A in AECOPD may thus be beneficial to reduce neutrophil recruitment to the airways.

BET Bromodomain Proteins Mediate Downstream Signaling Events following Growth Factor Stimulation in Human Lung Fibroblasts and Are Involved in Bleomycin-Induced Pulmonary Fibrosis

Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extra-terminal domain–containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor–responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.