Protective role of γδ T cells in cigarette smoke and influenza infection.

Airborne pathogens commonly trigger severe respiratory failure or death in smokers with lung disease. Cigarette smoking compromises the effectiveness of innate immunity against infections but the underlying mechanisms responsible for defective acquired immune responses in smokers remains less clear. We found that mice exposed to chronic cigarette smoke recovered poorly from primary Influenza A pneumonia with reduced type I and II interferons (IFNs) and viral-specific immunoglobulins, but recruited γδ T cells to the lungs that predominantly expressed interleukin 17A (IL-17A). Il-17a-/- mice exposed to smoke and infected with Influenza A also recruited γδ T cells to the lungs, but in contrast to wild-type mice, expressed increased IFNs, made protective influenza-specific antibodies, and recovered from infection. Depletion of IL-17A with blocking antibodies significantly increased T-bet expression in γδ T cells and improved recovery from acute Influenza A infection in air, but not smoke-exposed mice. In contrast, when exposed to smoke, γδ T cell deficient mice failed to mount an effective immune response to Influenza A and showed increased mortality. Our findings demonstrate a protective role for γδ T cells in smokers and suggest that smoke-induced increase in IL-17A inhibits the transcriptional programs required for their optimal anti-viral responses. Cigarette smoke induces IL-17A expression in the lungs and inhibits γδ T-cell-mediated protective anti-viral immune responses.

Cigarette Smoke and DNA Cleavage Promote Lung Inflammation and Emphysema.

Smoking-related lung diseases are among the most preventable and incurable ailments in the world. Smokers are at increased risk of developing chronic obstructive pulmonary disease that can be further complicated by emphysema and lung cancer. A subset of former smokers shows persistent lung inflammation and progressive loss of lung function, indicating a role for activation of acquired immunity in smoking-induced lung diseases. In addition to the well-established noxious effects of volatile compounds in cigarette smoke, incomplete combustion of tobacco generates nano-sized carbon black (nCB) that accumulate in lung myeloid dendritic cells and macrophages. Experimentally, intra-nasal instillation nCB can cause airway inflammation and emphysema in mice, underscoring their pathogenic role in inflammatory lung diseases. High throughput analyses of macrophages that have engulfed nCB reveal de novo activation of DNA repair enzymes, and histological studies provide evidence for DNA double-stranded breaks. Emphysematous lung myeloid dendritic cells that contain nCB express pro-inflammatory cytokines, and can efficiently differentiate naive CD4 T cells to interferon-g-secreting T helper 1 and interleukin 17A expressing cell subsets. Together these findings indicate that nCB accumulation in lung innate immune cells can initiate and sustain lung inflammation and promote emphysema development.

Activation of C3a receptor is required in cigarette smoke-mediated emphysema.

Exposure to cigarette smoke can initiate sterile inflammatory responses in the lung and activate myeloid dendritic cells (mDCs) that induce differentiation of T helper type 1 (Th1) and Th17 cells in the emphysematous lungs. Consumption of complement proteins increases in acute inflammation, but the contribution of complement protein 3 (C3) to chronic cigarette smoke-induced immune responses in the lung is not clear. Here, we show that following chronic exposure to cigarette smoke, C3-deficient (C3(-/-)) mice develop less emphysema and have fewer CD11b(+)CD11c(+) mDCs infiltrating the lungs as compared with wild-type mice. Proteolytic cleavage of C3 by neutrophil elastase releases C3a, which in turn increases the expression of its receptor (C3aR) on lung mDCs. Mice deficient in the C3aR (C3ar(-/-)) partially phenocopy the attenuated responses to chronic smoke observed in C3(-/-) mice. Consistent with a role for C3 in emphysema, C3 and its active fragments are deposited on the lung tissue of smokers with emphysema, and smoke-exposed mice. Together, these findings suggest a critical role for C3a through autocrine/paracrine induction of C3aR in the pathogenesis of cigarette smoke-induced sterile inflammation and provide new therapeutic targets for the treatment of emphysema.

Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13.

Although the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK- populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.

Multistrain influenza protection induced by a nanoparticulate mucosal immunotherapeutic.

All commercial influenza vaccines elicit antibody responses that protect against seasonal infection, but this approach is limited by the need for annual vaccine reformulation that precludes efficient responses against epidemic and pandemic disease. In this study we describe a novel vaccination approach in which a nanoparticulate, liposome-based agent containing short, highly conserved influenza-derived peptides is delivered to the respiratory tract to elicit potent innate and selective T cell-based adaptive immune responses. Prepared without virus-specific peptides, mucosal immunostimulatory therapeutic (MIT) provided robust, but short-lived, protection against multiple, highly lethal strains of influenza in mice of diverse genetic backgrounds. MIT prepared with three highly conserved epitopes that elicited virus-specific memory T-cell responses but not neutralizing antibodies, termed MITpep, provided equivalent, but more durable, protection relative to MIT. Alveolar macrophages were more important than dendritic cells in determining the protective efficacy of MIT, which induced both canonical and non-canonical antiviral immune pathways. Through activation of airway mucosal innate and highly specific T-cell responses, MIT and MITpep represent novel approaches to antiviral protection that offer the possibility of universal protection against epidemic and pandemic influenza.

Link between allergic asthma and airway mucosal infection suggested by proteinase-secreting household fungi.

Active fungal proteinases are powerful allergens that induce experimental allergic lung disease strongly resembling atopic asthma, but the precise relationship between proteinases and asthma remains unknown. Here, we analyzed dust collected from the homes of asthmatic children for the presence and sources of active proteinases to further explore the relationship between active proteinases, atopy, and asthma. Active proteinases were present in all houses and many were derived from fungi, especially Aspergillus niger. Proteinase-active dust extracts were alone insufficient to initiate asthma-like disease in mice, but conidia of A. niger readily established a contained airway mucosal infection, allergic lung disease, and atopy to an innocuous bystander antigen. Proteinase produced by A. niger enhanced fungal clearance from lung and was required for robust allergic disease. Interleukin 13 (IL-13) and IL-5 were required for optimal clearance of lung fungal infection and eosinophils showed potent anti-fungal activity in vitro. Thus, asthma and atopy may both represent a protective response against contained airway infection due to ubiquitous proteinase-producing fungi.

Cutting edge: the absence of C3 demonstrates a role for complement in Th2 effector functions in a murine model of pulmonary allergy.

Asthma is a chronic disease of the lung resulting from airway obstruction. Although the initiating causes are not entirely clear, the airway inflammation in asthma is associated with Th2 lymphocytes and their cytokines, particularly IL-4, which play a prominent role in this disease by regulating airway hyperresponsiveness, eosinophil activation, and IgE synthesis. Historically, complement was not thought to contribute to the pathogenesis of asthma. However, using C3-deficient mice in an allergen-induced model of pulmonary allergy, we demonstrate that complement may impact key features of this disease. When challenged with allergen, mice deficient in C3 exhibit diminished airway hyperresponsiveness and lung eosinophilia. Furthermore, these mice also have dramatically reduced numbers of IL-4-producing cells and attenuated Ag-specific IgE and IgG1 responses. Collectively, these results demonstrate that C3-deficient mice have significantly altered allergic lung responses and indicate a role for the complement system in promoting Th2 effector functions in asthma.

Selective aspects of the insulin resistance syndrome.

There is increasing recognition of new features in the insulin resistance syndrome and its association with new disease states or treatment modalities. Recent additions to the list of features in the insulin resistance syndrome include elevated non-esterified fatty acids, abnormalities in visceral fat metabolism, elevated uric acid, elevated hematocrit, endothelial dysfunction, abnormalities in glucocorticoids, and differences in the phenotypic expression of the syndrome between men and women. A critical factor that may be inherent in the syndrome is the distribution and metabolism of visceral fat. This finding is also accompanied by the recognition of the role of non-esterified fatty acids as a cause of many of the risk factors in the insulin resistance syndrome. Elevated non-esterified fatty acids contribute to hypertension, glucose intolerance and increased arteriosclerosis. Elevated cortisol levels and disrupted metabolism, as well as abnormalities in the hypothalamic-pituitary-adrenal axis are seen in the insulin resistance syndrome. In women, adipose cells express fewer glucocorticoid receptors and less of the enzyme that metabolizes cortisol, 11beta-hydroxysteroid dehydrogenase. Several inflammatory factors such as tumor necrosis factor-alpha may be an etiologic link in the risk found in the insulin resistance syndrome. Certain cases of the syndrome appear to be related to specific drug therapies (steroids, immunosuppressive agents and antiretroviral agents), as seen in transplant patients and HIV-infected individuals.

Sex-related differences in the insulin resistance syndrome.

In the insulin resistance (IR) syndrome, sex-specific differences have been reported. First, hypertension more often correlates with hyperinsulinemia in women than in men with the IR syndrome. In addition, salt sensitivity of blood pressure appears to be independent of the activity of the renin-angiotensin system in women, whereas in men there is a strong correlation between the two variables. Secondly, the dyslipidemia found in women with the IR syndrome is characterized by less postprandial plasma insulin, triglycerides, and fatty acid response to a standardized meal. However, this sex difference in lipids disappears after correction for visceral fat mass. Fat physiology and biochemistry differ between the two sexes. In women, adipose cells express less glucocorticoid receptors and less 11beta-hydroxysteroid dehydrogenase. In women visceral fat accumulation appears to be a constant feature of the IR syndrome but in men the syndrome can be present without central obesity. Lastly, during the reproductive years of women, the IR syndrome, such as in pre-eclampsia, may cause fetal growth retardation that has been proposed together with maternal malnutrition to be at the origin of the increased risk for impaired glucose tolerance, hyperinsulinemia, and hypertension in adult life. This gives yet another dimension to this disease in women since in essence they may ultimately transmit this syndrome to both sexes.

Protection from vascular risk in diabetic hypertension.

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia and associated with a high incidence of complications affecting both the microvascular and the macrovascular systems. Macrovascular disease affects the coronary arteries, the cerebral vessels, and the large peripheral arteries of the lower extremities. Microangiopathy affects the kidneys, eyes, and nerves. Both forms of complication are major causes of death and disability in diabetes. The precise pathophysiology of these vascular complications is becoming better understood, but specific treatment and prevention remain complex.

Baseline airway hyperreactivity in A/J mice is not mediated by cells of the adaptive immune system.

Human asthma is characterized by increased airway hyperreactivity to a variety of bronchoconstricting agents. Aberrant type 2 immune responses in the lung have been associated with airway hyperreactivity in both human asthma and in murine models of allergic airways disease. Despite their intrinsically elevated basal airway reactivity to smooth muscle constricting agents, A/J mice demonstrated no inherent inflammatory cell infiltration nor elevation of type 2 cytokines in the lung. Crossed bone marrow reconstitution experiments between A/J and MHC congenic B10.A mice revealed enhanced airway reactivity only in A/J recipients, irrespective of whether they had been reconstituted with A/J or B10. A hemopoietic cells. Further, A/J-derived bone marrow cells did not affect the reactivity of B10.A recipients. Although mice on RAG-deficient and IL-4-deficient backgrounds demonstrate substantial abrogation of allergen-induced airway hyperreactivity, these gene deletions had no impact on the elevated baseline reactivity when backcrossed onto A/J mice. Thus, in these mice, basal airway hyperreactivity is maintained independently of type 2 immunity induced by allergens.

Airway inflammation and remodeling in asthma.

An important advance in our understanding of the pathophysiology of asthma has been the discovery that airway inflammation is not confined to severe asthma but also characterizes mild and moderate asthma. Inflammation in asthma may be the result of a peculiar type of lymphocytic inflammation whereby Th2 lymphocytes secrete cytokines that orchestrate cellular inflammation and promote airway hyperresponsiveness. The term "airway remodeling" in asthma refers to structural changes that occur in conjunction with, or because of, chronic airway inflammation. Airway remodeling results in alterations in the airway epithelium, lamina propria, and submucosa, leading to thickening of the airway wall. The consequences of airway remodeling in asthma may include incompletely reversible airway narrowing, bronchial hyperresponsivenesss, airway edema, and mucus hypersecretion. Airway remodeling in asthma thus may predispose persons with asthma to asthma exacerbations and even death from airway obstruction caused by smooth muscle contraction, airway edema, and mucus plugging. Although much has been learned in the past 25 years about the pathophysiology of airway inflammation and airway remodeling in asthma, important questions remain about the relation between airway inflammation and remodeling, the natural history of airway remodeling, and the effects of current asthma treatments on remodeled airways.

Induction and regulation of the IgE response.

Immunoglobulin E (IgE) is believed to be one of the major mediators of immediate hypersensitivity reactions that underlie atopic conditions such as urticaria, seasonal allergy, asthma and anaphylaxis. Factors that control IgE production are therefore essential to the pathogenesis of these important afflictions. But a complete understanding of this topic is lacking, while new data have raised questions regarding the precise role of IgE in atopic disease. Evolving concepts of IgE production and elimination are likely to clarify the importance of IgE in health and disease.

IL-13 in allergy: home at last.

The immune response to allergic challenge is thought to include important effects mediated by cytokines. Of considerable interest to immunologists and parasitologists is the distinction between two closely related cytokines, IL-4 and IL-13. Although overlapping functions are inevitable, studies over the past year reveal a distinct role for IL-13 in mediating the physiologic response to a diverse array of allergens and parasites.

Obesity, hypertension, and sympathetic nervous system activity.

Several epidemiologic studies have shown that obesity represents an independent risk factor for the development of cardiovascular diseases, including hypertension, myocardial ischemic disease, and cardiac arrhythmias. One of the most appealing concepts in obesity-related hypertension is that a specific etiology can be identified. There is now substantial evidence that human obesity is characterized by abnormalities in sympathetic cardiovascular control. The application of sensitive techniques to assess sympathetic nervous system (SNS) activity in humans, including catecholamine levels, norepinephrine (NE) spillover techniques, and microneurography have furthered this concept. Catecholamine levels in obesity have been conflicting, with high, normal, and low levels reported. However, studies examining weight loss have found that the fall in blood pressure (BP) was highly correlated with reductions in plasma NE. Examination of NE spillover in obesity has shown regional overactivity in the kidneys. High renal SNS activity could lead to sodium retention and abnormal glomerular hemodynamics that could raise BP. Microneurography, which determines muscle sympathetic outflow, has shown consistent elevation in obesity, but no difference between normotensive and hypertensive obesity. However, the hyperinsulinemia of obesity may act in concert with the SNS to elevate BP, as the combination of the two seems to produce vascular constriction. Leptin also has several cardiovascular actions that may contribute to BP regulation. Epidemiologic studies also found that SNS activity predicts hypertension in obese subjects.

Construction of polycompetitors for competitive PCR.

Many different protocols are now available for competitive polymerase chain reaction (PCR) and most rely on the use of a mimic or competitor that serves as a reference for quantitation (1-4). The success (or failure) of all these protocols is critically dependent on the design, construction, and utilization of these constructs. This protocol provides detailed instructions for developing individual mimics, or competitors, for use in competitive PCR reactions. Individual competitors can be joined together in logical order in one plasmid, producing a single reagent, or polycompetitor, with multiple specificity. Although the protocol has been used successfully in producing cytokine polycompetitors, for both human and mouse (5), it should work well for almost any molecule of interest, provided sequence information is available. If a polycompetitor is to be synthesized, careful planning is especially required for a trouble-free outcome. Detailed restriction-endonuclease maps of the cloning vectors and PCR products to be cloned must be used in the design of primers and to plan appropriate strategies for incorporation of individual competitor constructs. Although many different cloning vectors may be used, in order not to be too general, this protocol provides detailed information using a commercially available vector, pGEM 11Z, and steps used in the construction of a specific polycompetitor, the human polycompetitor for T-cell cytokines, pDC10. The general principles, however, are applicable to the construction of polycom-petitors for any genes, using many different commercially available vectors.

Requirement for IL-13 independently of IL-4 in experimental asthma.

The pathogenesis of asthma reflects, in part, the activity of T cell cytokines. Murine models support participation of interleukin-4 (IL-4) and the IL-4 receptor in asthma. Selective neutralization of IL-13, a cytokine related to IL-4 that also binds to the alpha chain of the IL-4 receptor, ameliorated the asthma phenotype, including airway hyperresponsiveness, eosinophil recruitment, and mucus overproduction. Administration of either IL-13 or IL-4 conferred an asthma-like phenotype to nonimmunized T cell-deficient mice by an IL-4 receptor alpha chain-dependent pathway. This pathway may underlie the genetic associations of asthma with both the human 5q31 locus and the IL-4 receptor.

Requirements for allergen-induced airway hyperreactivity in T and B cell-deficient mice.

BACKGROUND: The pathogenesis of asthma is believed to reflect antigen-induced airway inflammation leading to the recruitment of eosinophils and activation of mast cells through cell-associated IgE. Controversies persist however, regarding the relative importance of different pathogenic cells and effector molecules. MATERIALS AND METHODS: A variety of gene-targeted mice were examined for the induction of cholinergic airway hyperresponsiveness (AH), allergic airway inflammation, mucus production, and serum IgE reactivity following intratracheal challenge with a potent allergen. AH was determined using whole-body plethysmography following acetylcholine challenge. Where possible, results were confirmed using neutralizing antibodies and cell-specific reconstitution of immune deficient mice. RESULTS: T and B cell-deficient, recombinase-activating-gene-deficient mice (RAG -/-) failed to develop significant allergic inflammation and AH following allergen challenge. Reconstitution of RAG -/- mice with CD4+ T cells alone was sufficient to restore allergen-induced AH, allergic inflammation, and goblet cell hyperplasia, but not IgE reactivity. Sensitized B cell-deficient mice also developed airway hyperreactivity and lung inflammation comparable to that of wild-type animals, confirming that antibodies were dispensable. Treatment with neutralizing anti-IL-4 antibody or sensitization of IL-4-deficient mice resulted in loss of airway hyperreactivity, whereas treatment with anti-IL-5 antibody or sensitization of IL-5-deficient mice had no effect. CONCLUSIONS: In mice, CD4+ T cells are alone sufficient to mediate many of the pathognomonic changes that occur in human asthma by a mechanism dependent upon IL-4, but independent of IL-5, IgE, or both. Clarification of the role played by CD4+ T cells is likely to stimulate important therapeutic advances in treatment of asthma.