A method to enable the investigation of murine bronchial immune cells, their cytokines and mediators

Innovative therapies for severe lung diseases (such as allergic and chronic asthma, chronic obstructive pulmonary disease or any type of lung cancer) require a detailed understanding of the cellular and immune processes in the lung. This protocol details a method to obtain the immune cells of the bronchi as well as the cytokines and mediators produced by these cells for further investigation. The broncho-alveolar lavage fluid (BALF) is taken by injecting physiological solution through the tracheal tube into the murine airways and carefully regained by winding up the connected syringe. After centrifugation, the resulting BALF supernatant can be stored for detection of cytokines or other mediators by enzyme-linked immunosorbent assay or other methods; the resuspended cell pellet can also be used for flow cytometric analyses, to check cell viability and the level of apoptosis, as well as other applications. In addition, CD4+ T cells isolated from wild-type and genetically modified mice alone or along with other immunologically important cells such as T regulatory cells, which can be used to reconstitute immunodeficient mice, may be retrieved from the airways with this method. This protocol can be completed within 35 min.     

The role of Th1/Th2 polarization in mucosal immunity

Mucosal immunity relies on the delicate balance between antigen responsiveness and tolerance. The polarization of T helper cells plays a key role in maintaining or disrupting this equilibrium.     

Universal influenza B vaccine based on the maturational cleavage site of the hemagglutinin precursor

Conventional influenza vaccines can prevent infection, but their efficacy depends on the degree of antigenic "match" between the strains used for vaccine preparation and those circulating in the population. A universal influenza vaccine based on invariant regions of the virus, able to provide broadly cross-reactive protection, without requiring continuous manufacturing update, would solve a major medical need. Since the temporal and geographical dominance of the influenza virus type and/or subtype (A/H3, A/H1, or B) cannot yet be predicted, a universal vaccine, like the vaccines currently in use, should include both type A and type B influenza virus components. However, while encouraging preclinical data are available for influenza A virus, no candidate universal vaccine is available for influenza B virus. We show here that a peptide conjugate vaccine, based on the highly conserved maturational cleavage site of the HA(0) precursor of the influenza B virus hemagglutinin, can elicit a protective immune response against lethal challenge with viruses belonging to either one of the representative, non-antigenically cross-reactive influenza B virus lineages. We demonstrate that protection by the HA(0) vaccine is mediated by antibodies, probably through effector mechanisms, and that a major part of the protective response targets the most conserved region of HA(0), the P1 residue of the scissile bond and the fusion peptide domain. In addition, we present preliminary evidence that the approach can be extended to influenza A virus, although the equivalent HA(0) conjugate is not as efficacious as for influenza B virus.     

IL-2 receptor beta-chain signaling controls immunosuppressive CD4+ T cells in the draining lymph nodes and lung during allergic airway inflammation in vivo

IL-2 influences both survival and differentiation of CD4(+) T effector and regulatory T cells. We studied the effect of i.n. administration of Abs against the alpha- and the beta-chains of the IL-2R in a murine model of allergic asthma. Blockade of the beta- but not the alpha-chain of the IL-2R after allergen challenge led to a significant reduction of airway hyperresponsiveness. Although both treatments led to reduction of lung inflammation, IL-2 signaling, STAT-5 phosphorylation, and Th2-type cytokine production (IL-4 and IL-5) by lung T cells, IL-13 production and CD4(+) T cell survival were solely inhibited by the blockade of the IL-2R beta-chain. Moreover, local blockade of the common IL-2R/IL-15R beta-chain reduced NK cell number and IL-2 production by lung CD4(+)CD25(+) and CD4(+)CD25(-) T cells while inducing IL-10- and TGF-beta-producing CD4(+) T cells in the lung. This cytokine milieu was associated with reduced CD4(+) T cell proliferation in the draining lymph nodes. Thus, local blockade of the beta-chain of the IL-2R restored an immunosuppressive cytokine milieu in the lung that ameliorated both inflammation and airway hyperresponsiveness in experimental allergic asthma. These findings provide novel insights into the functional role of IL-2 signaling in experimental asthma and suggest that blockade of the IL-2R beta-chain might be useful for therapy of allergic asthma in humans.     

DPP‐IV‐resistant,long‐acting oxyntomodulin derivatives

Obesity is one of the major risk factors for type 2 diabetes, and the development of agents, that can simultaneously achieve glucose control and weight loss, is being actively pursued. Therapies based on peptide mimetics of the gut hormone glucagon‐like peptide 1 (GLP‐1) are rapidly gaining favor, due to their ability to increase insulin secretion in a strictly glucose‐dependent manner, with little or no risk of hypoglycemia, and to their additional benefit of causing a modest, but durable weight loss. Oxyntomodulin (OXM), a 37‐amino acid peptide hormone of the glucagon (GCG) family with dual agonistic activity on both the GLP‐1 (GLP1R) and the GCG (GCGR) receptors, has been shown to reduce food intake and body weight in humans, with a lower incidence of treatment‐associated nausea than GLP‐1 mimetics. As for other peptide hormones, its clinical application is limited by the short circulatory half‐life, a major component of which is cleavage by the enzyme dipeptidyl peptidase IV (DPP‐IV). SAR studies on OXM, described herein, led to the identification of molecules resistant to DPP‐IV degradation, with increased potency as compared to the natural hormone. Analogs derivatized with a cholesterol moiety display increased duration of action in vivo. Moreover, we identified a single substitution which can change the OXM pharmacological profile from a dual GLP1R/GCGR agonist to a selective GLP1R agonist. The latter finding enabled studies, described in detail in a separate study (Pocai A, Carrington PE, Adams JR, Wright M, Eiermann G, Zhu L, Du X, Petrov A, Lassman ME, Jiang G, Liu F, Miller C, Tota LM, Zhou G, Zhang X, Sountis MM, Santoprete A, Capitò E, Chicchi GG, Thornberry N, Bianchi E, Pessi A, Marsh DJ, SinhaRoy R. Glucagon‐like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 2009; 58: 2258–2266), which highlight the potential of GLP1R/GCGR dual agonists as a potentially superior class of therapeutics over the pure GLP1R agonists currently in clinical use. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

IL-6 signaling in autoimmunity, chronic inflammation and inflammation-associated cancer

IL-6 activates various cell types carrying the membrane bound IL-6R (classical IL-6 signaling) as well as IL-6R(-) gp130(+) cells via the soluble IL-6R (IL-6 trans-signaling). IL-6 signaling plays a pivotal role in controlling the differentiation and activation of T lymphocytes by inducing the Jak/STAT-3 and the Ras/Erk/C/EBP pathways. In particular, IL-6 modulates the resistance of T cells against apoptosis, induces activation of T helper cells and controls the balance between regulatory T cells and Th17 cells. Importantly, recent findings suggest that blockade of IL-6 signaling is effective in treating experimental models of autoimmune and chronic inflammatory diseases such as inflammatory bowel diseases, diabetes, multiple sclerosis, asthma and rheumatoid arthritis as well as models of inflammation-associated cancer. Thus, anti-IL-6/anti-IL-6R strategies emerge as promising novel approaches for therapy of inflammatory diseases in humans. In this review article, we discuss the latest findings on the role of IL-6 in experimental models of autoimmunity and cancer, as well as clinical perspectives.     

Isolation of CD4+ T cells from murine lungs: a method to analyze ongoing immune responses in the lung

The regulation of the cellular immune response in lung diseases is not yet fully understood. Isolating different subsets of immune cells directly from the lung is therefore an indispensable method of gaining detailed knowledge on the function of these cells in this organ. This protocol describes a method of isolating and magnetically labeling CD4+ lung T cells, which are then loaded and retained on the column while all other cells run through it (positive selection). The yield of this isolation is approximately 5 x 10(5) to 1.5 x 10(6) CD4+ cells from a murine lung. These cells can be further investigated by several methods such as flow cytometry, western blot analysis, RT-PCR, immunostaining and ELISA. In addition, lung CD4+ T cells alone or along with other immunologically important cells such as CD8+ T cells and T regulatory cells can be adoptively transferred into immune-deficient mice, and can influence important local parameters. This protocol can be completed in approximately 4 h 20 min.