Gordon J. Gaschler

Bacteria challenge in smoke-exposed mice exacerbates inflammation and skews the inflammatory profile.

RATIONALE The pathogenesis of chronic obstructive pulmonary disease is associated with acute episodes of bacterial exacerbations. The most commonly isolated bacteria during episodes of exacerbation is nontypeable Haemophilus influenzae (NTHI). OBJECTIVES In this study, we investigated the in vivo consequences of cigarette smoke exposure on the inflammatory response to an NTHI challenge. METHODS C57BL/6 and BALB/c mice were exposed to cigarette smoke for 8 weeks and subsequently challenged intranasally with NTHI. MEASUREMENTS AND MAIN RESULTS We observed increased pulmonary inflammation and lung damage in cigarette smoke-exposed NTHI-challenged mice as compared with control NTHI-challenged mice. Furthermore, although NTHI challenge in control mice was marked by increases in tumor necrosis factor-alpha, IL-6, MIP-2, and KC/GROalpha, NTHI challenge in cigarette smoke-exposed mice led to a prominent up-regulation of a different subset of inflammatory mediators, most notably MCP-1, -3, and -5, IP-10, and MIP-1gamma. This skewed inflammatory mediator expression was also observed after ex vivo NTHI stimulation of alveolar macrophages, signifying their importance to this altered response. Importantly, corticosteroids attenuated inflammation after NTHI challenge in both cigarette smoke-exposed and control mice; however, this was associated with significantly increased bacterial burden. CONCLUSIONS Collectively, these data suggest that cigarette smoke exacerbates the inflammatory response to a bacterial challenge via skewed inflammatory mediator expression.

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.

Critical Negative Regulation of Type 1 T Cell Immunity and Immunopathology by Signaling Adaptor DAP12 during Intracellular Infection

Transmembrane signaling adaptor DAP12 has increasingly been recognized for its important role in innate responses. However, its role in the regulation of antimicrobial T cell responses has remained unknown. In our current study, we have examined host defense, T cell responses, and tissue immunopathology in models of intracellular infection established in wild-type and DAP12-deficient mice. During mycobacterial infection, lack of DAP12 leads to pronounced proinflammatory and Th1 cytokine responses, overactivation of Ag-specific CD4 and CD8 T cells of type 1 phenotype, and heightened immunopathology both in the lung and lymphoid organs. DAP12-deficient airway APC display enhanced NF-κB activation and cytokine responses upon TLR stimulation or mycobacterial infection in vitro. Of importance, adoptive transfer of Ag-loaded DAP12-deficient APC alone could lead to overactivation of transferred transgenic or endogenous wild-type T cells in vivo. We have further found that the immune regulatory role by DAP12 is not restricted only to intracellular bacterial infection, since lack of this molecule also leads to uncontrolled type 1 T cell activation and severe immunopathology and tissue injury during intracellular viral infection. Our study thus identifies DAP12 as an important novel immune regulatory molecule that acts, via APC, to control the level of antimicrobial type 1 T cell activation and immunopathology.

Mechanisms of clearance of nontypeable Haemophilus influenzae from cigarette smoke-exposed mouse lungs

Inflammation is prevalent in all stages of chronic obstructive pulmonary disease, and, furthermore, individuals undergo periods of exacerbation, during which pulmonary inflammation increases, often a result of bacterial infection. The present study investigates the in vivo consequences of cigarette smoke exposure on bacterial challenge with nontypeable Haemophilus influenzae (NTHi). BALB/c and C57 black 6 (C57BL/6) mice were exposed to cigarette smoke once or twice daily for a total period of 8 weeks. Exacerbated inflammation was observed in cigarette smoke-exposed compared to room-air-exposed mice following challenge with live or heat-inactivated NTHi. Accelerated clearance of live NTHi from cigarette smoke-exposed mice was independent of the establishment of chronic inflammation or direct toxic effects of cigarette smoke components on bacteria. Mechanistically, a cell-free factor in the bronchoalveolar lavage fluid contributed to accelerated clearance following passive transfer to naive mice. Further investigation demonstrated increased titres of immunoglobulin A in the bronchoalveolar lavage fluid, but not the blood, of cigarette smoke-exposed mice, including increased titres of NTHi-specific immunoglobulin A, whereas heavy chain joining element (JH)-/- B-cell-deficient cigarette smoke-exposed mice did not demonstrate decreased bacterial burden following challenge. The present results demonstrate that cigarette smoke exposure results in exacerbated inflammation following challenge with NTHi, as well as increased titres of antibodies that contribute to bacterial clearance.

Impact of cigarette smoke on T and B cell responsiveness

Although its direct effects cannot be discounted, tobaccos effects on the immune system have been proposed to play a key role in mediating its deleterious health impact. Studies in rats using high levels of smoke exposure have suggested that tobacco smoke exhausts cellular signal transduction cascades, making lymphocytes unresponsive to stimulation. In the present study, we show that purified B or T cells, and total lymphocytes from the lungs, lymph nodes and spleens of smoke-exposed mice fluxed calcium, proliferated, and secreted immunoglobulin or IFN-gamma similarly to control mice when stimulated with ligands including anti-IgM, and anti-CD3. Importantly, we recapitulated these findings in PBMCs from human smokers; cells from long-term smokers and never-smokers proliferated equivalently when stimulated ex vivo. Previous reports of lymphocyte unresponsiveness in rats are inconsistent with these findings, and may reflect a phenomenon observed only at levels of smoke exposure well above those seen in actual human smokers.

Lung Epithelial CCAAT/Enhancer-binding Protein-β Is Necessary for the Integrity of Inflammatory Responses to Cigarette Smoke

RATIONALE Cigarette smoke is the major cause of chronic obstructive pulmonary disease and lung cancer. The mechanisms by which smoking induces pulmonary dysfunction are complex, involving stress from toxic components and inflammatory responses. Although CCCAAT/enhancer-binding protein (C/EBP)-β is known as a key intracellular regulator of inflammatory signaling, its role in pulmonary inflammation has not been established. OBJECTIVES To characterize the role of C/EBPβ in the airway epithelial response to cigarette smoke. METHODS mRNA expression in the airway epithelium of current, former, and never-smokers, and in in vitro cigarette smoke extract-treated primary human airway epithelial cells, was analyzed by microarray and quantitative real-time polymerase chain reaction, respectively. Mice with lung epithelial-specific inactivation of C/EBPβ were generated and exposed to cigarette smoke for 4 or 11 days. Lung histology, bronchoalveolar lavage cell differentials, and expression of inflammatory and innate immune mediators in the lungs were assessed. MEASUREMENTS AND MAIN RESULTS C/EBPβ was significantly down-regulated in the airway epithelium of both current and former smokers compared with never-smokers, and in cigarette smoke-treated primary human airway epithelial cells in vitro. Cigarette smoke-exposed mice with a lung epithelial-specific inactivation of C/EBPβ displayed blunted respiratory neutrophil influx and compromised induction of neutrophil chemoattractants growth-regulated oncogene-α, macrophage inflammatory protein-1γ, granulocyte colony-stimulating factor, and serum amyloid A 3 and proinflammatory cytokines tumor necrosis factor-α and interleukin-1β, compared with smoke-exposed controls. Inhibition of C/EBPβ in human airway cells in vitro caused a similarly compromised response to smoke. CONCLUSION Our data suggest a previously unknown role for C/EBPβ and the airway epithelium in mediating inflammatory and innate immune responses to cigarette smoke.

Impact of Cigarette Smoke on the Human and Mouse Lungs : A Gene-Expression Comparison Study

Cigarette smoke is well known for its adverse effects on human health, especially on the lungs. Basic research is essential to identify the mechanisms involved in the development of cigarette smoke-related diseases, but translation of new findings from pre-clinical models to the clinic remains difficult. In the present study, we aimed at comparing the gene expression signature between the lungs of human smokers and mice exposed to cigarette smoke to identify the similarities and differences. Using human and mouse whole-genome gene expression arrays, changes in gene expression, signaling pathways and biological functions were assessed. We found that genes significantly modulated by cigarette smoke in humans were enriched for genes modulated by cigarette smoke in mice, suggesting a similar response of both species. Sixteen smoking-induced genes were in common between humans and mice including six newly reported to be modulated by cigarette smoke. In addition, we identified a new conserved pulmonary response to cigarette smoke in the induction of phospholipid metabolism/degradation pathways. Finally, the majority of biological functions modulated by cigarette smoke in humans were also affected in mice. Altogether, the present study provides information on similarities and differences in lung gene expression response to cigarette smoke that exist between human and mouse. Our results foster the idea that animal models should be used to study the involvement of pathways rather than single genes in human diseases.

Dysregulated Macrophage-Inflammatory Protein-2 Expression Drives Illness in Bacterial Superinfection of Influenza

Influenza virus infection is a leading cause of death and disability throughout the world. Influenza-infected hosts are vulnerable to secondary bacterial infection, however, and an ensuing bacterial pneumonia is actually the predominant cause of influenza-attributed deaths during pandemics. A number of mechanisms have been proposed by which influenza may predispose to superinfection with an unrelated or heterologous pathogen, but the subsequent interaction between the host, virus, and bacteria remains an understudied area. In this study, we develop and examine a novel model of heterologous pulmonary infection in which an otherwise subclinical Bordetella parapertussis infection synergizes with an influenza virus infection to yield a life-threatening secondary pneumonia. Despite a profound pulmonary inflammatory response and unaltered viral clearance, bacterial clearance was significantly impaired in heterologously infected mice. No deficits were observed in pulmonary or systemic adaptive immune responses or the viability or function of infiltrating inflammatory cells to explain this phenomenon, and we provide evidence that the onset of severe pulmonary inflammation actually precedes the increased bacterial burden, suggesting that exacerbated inflammation is independent of bacterial burden. To that end, neutralization of the ELR+ inflammatory chemokine MIP-2 (CXCL2/GRO-β) attenuated the inflammation, weight loss, and clinical presentation of heterologously infected mice without impacting bacterial burden. These data suggest that pulmonary inflammation, rather than pathogen burden, is the key threat during bacterial superinfection of influenza and that selective chemokine antagonists may be a novel therapeutic intervention in cases of bacterial superinfection of influenza.

Animal Models of Chronic Obstructive Pulmonary Disease Exacerbations

Modeling acute exacerbations of chronic obstructive pulmonary disease (AECOPD) in animals has proven challenging due to the clinical and pathological complexity of the underlying disease. This has hindered the progress in understanding the cellular and molecular mechanisms that lie beneath AECOPD. In this chapter, we will address modeling possibilities of AECOPD that may be drawn from the current knowledge of factors that cause exacerbations. Importantly, since it is widely accepted that the most common causes of AECOPD are viral and bacterial infections, animal models of AECOPD should incorporate both the causative agents of exacerbation: viruses and bacteria. However, other factors that are also believed to determine both progression of COPD, as well as the frequency and severity of AECOPD, such as proteolytic enzymes, cigarette smoke or other noxious stimuli must also be considered. Such animal models will provide mechanistic insight into the etiology of AECOPD and will prove invaluable in furthering our understanding of key events in disease pathogenesis.