Dietary wheat amylase trypsin inhibitors exacerbate murine allergic airway inflammation.

BACKGROUND: Wheat amylase trypsin inhibitors (ATI) are dietary non-gluten proteins that activate the toll-like receptor 4 on myeloid cells, promoting intestinal inflammation. AIM OF THE STUDY: We investigated the effects of dietary ATI on experimental allergic airway inflammation. METHODS: Mice on a gluten and ATI-free diet (GAFD), sensitized with PBS or ovalbumin (OVA) and challenged with OVA, were compared to mice on a commercial standard chow, a gluten diet naturally containing ~ 0.75% of protein as ATI (G+AD), a gluten diet containing ~ 0.19% of protein as ATI (G-AD) and a GAFD with 1% of protein as ATI (AD). Airway hyperreactivity (AHR), inflammation in bronchoalveolar lavage (BAL) and pulmonary tissue sections were analyzed. Allergic sensitization was assessed ex vivo via proliferation of OVA-stimulated splenocytes. RESULTS: Mice on a GAFD sensitized with PBS did not develop AHR after local provocation with methacholine. Mice on a GAFD or on a G-AD and sensitized with OVA developed milder AHR compared to mice fed a G+AD or an AD. The increased AHR was paralleled by increased BAL eosinophils, IL-5 and IL-13 production, and an enhanced ex vivo splenocyte activation in the ATI-fed groups. CONCLUSIONS: Dietary ATI enhance allergic airway inflammation in OVA-challenged mice, while an ATI-free or ATI-reduced diet has a protective effect on AHR. Nutritional wheat ATI, activators of intestinal myeloid cells, may be clinically relevant adjuvants to allergic airway inflammation.

A key regulatory role of the transcription factor NFATc2 in bronchial adenocarcinoma via CD8+ T lymphocytes.

The Ca(2+)-regulated calcineurin/nuclear factor of activated T cells (NFAT) cascade controls alternative pathways of T-cell activation and peripheral tolerance. Here, we describe reduction of NFATc2 mRNA expression in the lungs of patients with bronchial adenocarcinoma. In a murine model of bronchoalveolar adenocarcinoma, mice lacking NFATc2 developed more and larger solid tumors than wild-type littermates. The extent of central tumor necrosis was decreased in the tumors in NFATc2((-/-)) mice, and this finding was associated with reduced tumor necrosis factor-alpha and interleukin-2 (IL-2) production by CD8(+) T cells. Adoptive transfer of CD8(+) T cells of NFATc2((-/-)) mice induced transforming growth factor-beta(1) in the airways of recipient mice, thus supporting CD4(+)CD25(+)Foxp-3(+)glucocorticoid-induced tumor necrosis factor receptor (GITR)(+) regulatory T (T(reg)) cell survival. Finally, engagement of GITR in NFATc2((-/-)) mice induced IFN-gamma levels in the airways, reversed the suppression by T(reg) cells, and costimulated effector CD4(+)CD25(+) (IL-2Ralpha) and memory CD4(+)CD127(+) (IL-7Ralpha) T cells, resulting in abrogation of carcinoma progression. Agonistic signaling through GITR, in the absence of NFATc2, thus emerges as a novel possible strategy for the treatment of human bronchial adenocarcinoma in the absence of NFATc2 by enhancing IL-2Ralpha(+) effector and IL-7Ralpha(+) memory-expressing T cells.

Immunosurveillance of lung melanoma metastasis in EBI-3-deficient mice mediated by CD8+ T cells.

EBV-induced gene 3 (EBI-3) codes for a soluble type I receptor homologous to the p40 subunit of IL-12 that is expressed by APCs following activation. In this study, we assessed the role of EBI-3 in a model of lung melanoma metastasis. Intravenous injection of the B16-F10 cell line resulted in a significant reduction of lung tumor metastasis in EBI-3(-/-) recipient mice compared with wild-type mice. The immunological finding accompanying this effect was the expansion of a newly described cell subset called IFN-gamma producing killer dendritic cells associated with CD8(+) T cell responses in the lung of EBI-3(-/-) mice including IFN-gamma release and TNF-alpha-induced programmed tumor cell death. Depletion of CD8(+) T cells as well as targeting T-bet abrogated the protective effects of EBI-3 deficiency on lung melanoma metastases. Finally, adoptive transfer of EBI-3(-/-) CD8(+) T cells into tumor bearing wild-type mice inhibited lung metastasis in recipient mice. Taken together, these data demonstrate that targeting EBI-3 leads to a T-bet-mediated antitumor CD8(+) T cell responses in the lung.

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.

Protective role of nuclear factor of activated T cells 2 in CD8+ long-lived memory T cells in an allergy model.

BACKGROUND: The transcriptional regulation of cytokines released and controlled by memory T cells is not well understood. Defective IFN-gamma production in allergic asthma correlates in human beings with the risk of wheezing in childhood. OBJECTIVE: To understand the role of the transcription factor nuclear factor of activated T cells 2 (NFATc2) in memory and effector T cells in the airways in experimental allergic asthma. METHODS: We used murine models of allergic asthma and adoptive cell transfer of fluorescence-activated sorted cells in a disease model. RESULTS: Mice lacking NFATc2 developed an increase in airwayhyperresponsiveness (AHR), remodeling, and serum IgE levelson ovalbumin sensitization. This phenotype was associated withCD81CD1222 T cells deficient in IFN-g production in theairways. The origin of this phenotype in NFATc2(2/2) mice wasrelated to an expanded population of lung CD81CD1221(IL-2Rb chain) CD127hi (IL-7 receptor [R] a chain1) long-livedmemory cells. Adoptive transfer of ovalbumin-specific CD81NFATc2(2/2) T cells enhanced the AHR generated byNFATc2(2/2) CD41 T cells in immunodeficient mice, increasedIL-17, and reduced IFN-g production in the reconstituted mice. Depletion of the memory CD81CD1221IL-7Rhigh T-cellpopulation corrected the defect in IFN-g production by lungNFATc2(2/2) CD81CD1222 cells and abrogated the increasedAHR observed in NFATc2(2/2) CD81 T-cell-reconstituted micewith a severe combined immunodeficiency disorder. CONCLUSION: Taken together, our results suggest that NFATc2 expression in long-lived memory CD8+ T cells controls IL-2 and IFN-gamma production in lung CD8+ T cells, which then limits TH17 and TH2 development in the airways during allergen challenge.

Lung CD11c+ cells from mice deficient in Epstein-Barr virus-induced gene 3 (EBI-3) prevent airway hyper-responsiveness in experimental asthma.

Epstein-Barr virus-induced gene (EBI)-3 codes for a soluble type 1 cytokine receptor homologous to the p40 subunit of IL-12 that is expressed by antigen-presenting cells following activation. Here, we analyzed the functional role of EBI-3 in a murine model of asthma associated with airway hyper-responsiveness (AHR) in ovalbumin-sensitized mice. Upon allergen challenge, EBI-3-/- mice showed less severe AHR, decreased numbers and degranulation of eosinophils and a significantly reduced number of VCAM-1+ cells in the lungs as compared to wild-type littermates. We thus analyzed lung CD11c+ cells before and after allergen challenge in these mice and found that before allergen challenge, lung CD11c+ cells isolated from EBI-3-/- mice express markers of a more plasmacytoid phenotype without releasing IFN-alpha as compared to those from wild-type littermates. Moreover, allergen challenge induced the development of myeloid CD11c+ cells in the lungs of EBI-3-/- mice, which released increased amounts of IL-10 and IL-12 while not expressing IFN-alpha. Finally, inhibition of EBI-3 expression in lung DC could prevent AHR in adoptive transfer studies by suppressing mediator release of effector cells into the airways. These results indicate a novel role for EBI-3 in controlling local immune responses in the lungs in experimental asthma.

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.

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.

A novel type of co-chaperone mediates transmembrane recruitment of DnaK-like chaperones to ribosomes.

Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes. This pancreatic DnaJ-like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK-like proteins, such as BiP. The pancreatic endoplasmic reticulum contains a second DnaJ-like membrane protein, which had been termed Mtj1p in mouse. Mtj1p is present in pancreatic microsomes at a lower concentration than Sec63p but has a higher affinity for BiP. In addition to a lumenal J-domain, Mtj1p contains a single transmembrane domain and a cytosolic domain which is in close contact with translating ribosomes and appears to have the ability to modulate translation. The interaction with ribosomes involves a highly charged region within the cytosolic domain of Mtj1p. We propose that Mtj1p represents a novel type of co-chaperone, mediating transmembrane recruitment of DnaK-like chaperones to ribosomes and, possibly, transmembrane signaling between ribosomes and DnaK-like chaperones of the endoplasmic reticulum.