Jake K. Nikota

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.

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.

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.

IL-1 Receptor Regulates microRNA-135b Expression in a Negative Feedback Mechanism during Cigarette Smoke–Induced Inflammation

Although microRNA-135b (miR-135b) is known to be associated with cancer, with recent work showing that it is massively induced in the pulmonary tissues of mice challenged with nanoparticles suggests a critical role for this microRNA in mediating inflammatory response. In this study, we investigated the expression and function of miR-135b in mice exposed to cigarette smoke or nontypeable Haemophilus influenzae (NTHi). Exposure to both cigarette smoke and NTHi elicited robust lung inflammation, but increased miR-135b expression was observed only in the lungs of cigarette smoke–exposed mice. Using IL-1R 1 knockout mice, we show that miR-135b expression is IL-1R1 dependent. A series of in vitro experiments confirmed the role of IL-1R1 in regulating miR-135b expression. In vitro activation of the IL-1R1 pathway in mouse embryonic fibroblast (NIH3T3) and lung epithelial (FE1) cells resulted in increased miR-135b, which was blocked by IL-1R1 antagonists or small interfering RNA–mediated silencing of IL-1R1 expression. Overexpression of mature miR-135b in NIH3T3 cells (pEGP-mmu-mir-135b) resulted in the suppression of endogenous levels of IL-1R1 expression. pEGP-mmu-miR-135b cells transiently transfected with luciferase reporter vector containing the 3′UTR of mouse IL-1R1 showed reduced luciferase activity. Finally, we demonstrate that miR-135b targets IL-1–stimulated activation of Caspase-1, the IL-1R1 downstream activator of IL-1β leading to suppressed synthesis of the active form of IL-1β protein. These results suggest that miR-135b expression during cigarette smoke–induced inflammation is regulated by IL-1R1 in a regulatory feedback mechanism to resolve inflammation.

Cigarette smoke-induced accumulation of lung dendritic cells is interleukin-1α-dependent in mice

BackgroundEvidence suggests that dendritic cells accumulate in the lungs of COPD patients and correlate with disease severity. We investigated the importance of IL-1R1 and its ligands IL-1α and β to dendritic cell accumulation and maturation in response to cigarette smoke exposure.MethodsMice were exposed to cigarette smoke using a whole body smoke exposure system. IL-1R1-, TLR4-, and IL-1α-deficient mice, as well as anti-IL-1α and anti-IL-1β blocking antibodies were used to study the importance of IL-1R1 and TLR4 to dendritic cell accumulation and activation.ResultsAcute and chronic cigarette smoke exposure led to increased frequency of lung dendritic cells. Accumulation and activation of dendritic cells was IL-1R1/IL-1α dependent, but TLR4- and IL-1β-independent. Corroborating the cellular data, expression of CCL20, a potent dendritic cells chemoattractant, was IL-1R1/IL-1α-dependent. Studies using IL-1R1 bone marrow-chimeric mice revealed the importance of IL-1R1 signaling on lung structural cells for CCL20 expression. Consistent with the importance of dendritic cells in T cell activation, we observed decreased CD4+ and CD8+ T cell activation in cigarette smoke-exposed IL-1R1-deficient mice.ConclusionOur findings convey the importance of IL-1R1/IL-1α to the recruitment and activation of dendritic cells in response to cigarette smoke exposure.

Cigarette Smoke Primes the Pulmonary Environment to IL-1α/CXCR-2–Dependent Nontypeable Haemophilus influenzae–Exacerbated Neutrophilia in Mice

Cigarette smoke has a broad impact on the mucosal environment with the ability to alter host defense mechanisms. Within the context of a bacterial infection, this altered host response is often accompanied by exacerbated cellular inflammation, characterized by increased neutrophilia. The current study investigated the mechanisms of neutrophil recruitment in a murine model of cigarette smoke exposure and, subsequently, a model of both cigarette smoke exposure and bacterial infection. We investigated the role of IL-1 signaling in neutrophil recruitment and found that cigarette smoke-induced neutrophilia was dependent on IL-1α produced by alveolar macrophages. In addition to being the crucial source of IL-1α, alveolar macrophages isolated from smoke-exposed mice were primed for excessive IL-1α production in response to bacterial ligands. To test the relevance of exaggerated IL-1α production in neutrophil recruitment, a model of cigarette smoke exposure and nontypeable Haemophilus influenzae infection was developed. Mice exposed to cigarette smoke elaborated an exacerbated CXCR2-dependent neutrophilia in response to nontypeable Haemophilus influenzae. Exacerbated neutrophilia was dependent on IL-1α priming of the pulmonary environment by cigarette smoke as exaggerated neutrophilia was dependent on IL-1 signaling. These data characterize a novel mechanism of cigarette smoke priming the lung mucosa toward greater IL-1–driven neutrophilic responses to bacteria, with a central role for the alveolar macrophage in this process.

Cigarette Smoke Differentially Affects Eosinophilia and Remodeling in a Model of House Dust Mite Asthma

Although a similar prevalence of smoking is evident among patients with asthma and the general population, little is known about the impact of cigarette smoke on the immune inflammatory processes elicited by common environmental allergens. We investigated the impact of exposure to cigarette smoke on house dust mite (HDM)-induced allergic airway inflammation and its consequences for tissue remodeling and lung physiology in mice. BALB/c mice received intranasal HDMs daily, 5 days per week, for 3 weeks to establish chronic airway inflammation. Subsequently, mice were concurrently exposed to HDMs plus cigarette smoke, 5 days per week, for 2 weeks (HDMs + smoke). We observed significantly attenuated eosinophilia in the bronchoalveolar lavage of mice exposed to HDMs + smoke, compared with animals exposed only to HDMs. A similar activation of CD4 T cells and expression of IL-5, IL-13, and transforming growth factor-β was observed between HDM-treated and HDM + smoke-treated animals. Consistent with an effect on eosinophil trafficking, HDMs + smoke exposure attenuated the HDM-induced expression of eotaxin-1 and vascular cell adhesion molecule-1, whereas the survival of eosinophils and the numbers of blood eosinophils were not affected. Exposure to cigarette smoke also reduced the activation of B cells and the concentrations of serum IgE. Although the production of mucus decreased, collagen deposition significantly increased in animals exposed to HDMs + smoke, compared with animals exposed only to HDMs. Although airway resistance was unaffected, tissue resistance was significantly decreased in mice exposed to HDMs + smoke. Our findings demonstrate that cigarette smoke affects eosinophil migration without affecting airway resistance or modifying Th2 cell adaptive immunity in a murine model of HDM-induced asthma.

Cigarette smoke-induced inflammation and respiratory host defense: Insights from animal models

While the devastating impact of tobacco on human health is well established, and efforts to reduce its prevalence are ongoing, over 1 billion people continue to smoke. Emerging evidence suggests that cigarette smoking distorts lung immune homeostasis, compromising respiratory host defense. Consequently, viral and bacterial agents are dealt with inefficiently and are associated with exaggerated immune inflammatory responses. In this article, we discuss mechanisms by which cigarette smoke elicits inflammatory processes and how smoking impacts respiratory host defense against viral and bacterial agents. Elucidating cigarette smokes impacts on lung immune homeostasis will contribute to our understanding of the pathogenesis of chronic obstructive pulmonary disease COPD.

Persistence of pulmonary tertiary lymphoid tissues and anti-nuclear antibodies following cessation of cigarette smoke exposure

Formation of pulmonary tertiary immune structures is a characteristic feature of advanced COPD. In the current study, we investigated the mechanisms of tertiary lymphoid tissue (TLT) formation in the lungs of cigarette smoke-exposed mice. We found that cigarette smoke exposure led to TLT formation that persisted following smoking cessation. TLTs consisted predominantly of IgM positive B cells, while plasma cells in close proximity to TLTs expressed IgM, IgG, and IgA. The presence of TLT formation was associated with anti-nuclear autoantibody (ANA) production that also persisted following smoking cessation. ANAs were observed in the lungs, but not the circulation of cigarette smoke-exposed mice. Similarly, we observed ANA in the sputum of COPD patients where levels correlated with disease severity and were refractory to steroid treatment. Both ANA production and TLT formation were dependent on interleukin-1 receptor 1 (IL-1R1) expression. Contrary to TLT and ANA, lung neutrophilia resolved following smoking cessation. These data suggest a differential regulation of innate and B cell-related immune inflammatory processes associated with cigarette smoke exposure. Moreover, our study further emphasizes the importance of interleukin-1 (IL-1) signaling pathways in cigarette smoke-related pulmonary pathogenesis.

Alterations in Skeletal Muscle Cell Homeostasis in a Mouse Model of Cigarette Smoke Exposure

Background Skeletal muscle dysfunction is common in chronic obstructive pulmonary disease (COPD), a disease mainly caused by chronic cigarette use. An important proportion of patients with COPD have decreased muscle mass, suggesting that chronic cigarette smoke exposure may interfere with skeletal muscle cellular equilibrium. Therefore, the main objective of this study was to investigate the kinetic of the effects that cigarette smoke exposure has on skeletal muscle cell signaling involved in protein homeostasis and to assess the reversibility of these effects. Methods A mouse model of cigarette smoke exposure was used to assess skeletal muscle changes. BALB/c mice were exposed to cigarette smoke or room air for 8 weeks, 24 weeks or 24 weeks followed by 60 days of cessation. The gastrocnemius and soleus muscles were collected and the activation state of key mediators involved in protein synthesis and degradation was assessed. Results Gastrocnemius and soleus were smaller in mice exposed to cigarette smoke for 8 and 24 weeks compared to room air exposed animals. Pro-degradation proteins were induced at the mRNA level after 8 and 24 weeks. Twenty-four weeks of cigarette smoke exposure induced pro-degradation proteins and reduced Akt phosphorylation and glycogen synthase kinase-3β quantity. A 60-day smoking cessation period reversed the cell signaling alterations induced by cigarette smoke exposure. Conclusions Repeated cigarette smoke exposure induces reversible muscle signaling alterations that are dependent on the duration of the cigarette smoke exposure. These results highlights a beneficial aspect associated with smoking cessation.